T cell modifying compounds and uses thereof

ABSTRACT

Methods and compositions for modifying T-cells in which PD1 and/or CTLA-4 is repressed and/or inactivated using fusion proteins such as artificial transcription factors and nucleases.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/051,244, filed Oct. 10, 2013, which claims the benefit ofU.S. Provisional Application No. 61/712,028, filed Oct. 10, 2012, thedisclosures of which are hereby incorporated by reference in theirentireties.

TECHNICAL FIELD

The present disclosure is in the fields of genome editing andtherapeutics.

BACKGROUND

Engineered nucleases, including zinc finger nucleases, TALENs and homingendonucleases designed to specifically bind to target DNA sites areuseful in genome engineering. For example, zinc finger nucleases (ZFNs)are proteins comprising engineered site-specific zinc fingers fused to anuclease domain. Such ZFNs and TALENs have been successfully used forgenome modification in a variety of different species. See, for example,United States Patent Publications 20030232410; 20050208489; 20050026157;20050064474; 20060188987; 20060063231; 20110301073; 20130177983;20130177960; 20150056705 and International Publication WO 07/014275, thedisclosures of which are incorporated by reference in their entiretiesfor all purposes. These engineered nucleases can create a double-strandbreak (DSB) at a specified nucleotide sequence, which increases thefrequency of homologous recombination at the targeted locus by more than1000-fold. Thus, engineered nucleases can be used to exploit thehomology-directed repair (HDR) system and facilitate targetedintegration of transgenes into the genome of cells. In addition, theinaccurate repair of a site-specific DSB by non-homologous end joining(NHEJ) can also result in gene disruption.

The programmed death receptor (PD1 or PD-1, also known as PDCD1) hasbeen shown to be involved in regulating the balance between T-cellactivation and T-cell tolerance in response to chronic antigens, and isencoded by one of a group of genes known as immunological checkpointgenes. The proteins encoded by these genes are involved in regulatingthe amplitude of immune responses. Upon T-cell activation, PD1expression is induced in T-cells. The ligands for the PD1 receptor arePD1 ligand (PDL1 also known as B7-H1 and CD272) and PDL2 (also known asB7-DC and CD273), and are normally expressed in antigen presentingcells. PD1-PDL (PD1 ligand) coupling causes deactivation of the T-celland is involved in inducing T-cell tolerance (see, Pardoll (2012) NatRev 12:252). During HIV1 infection, expression of PD1 has been found tobe increased in CD4+ T-cells, and PDL1 expression is increased in APCs,tipping the balance between T-cell inhibition and T-cell stimulationtowards T-cell inhibition (see Freeman et al (2006) J Exp Med203(10):2223-2227). It is thought that PD1 up-regulation is somehow tiedto T-cell exhaustion (defined as a progressive loss of key effectorfunctions) when T-cell dysfunction is observed in the presence ofchronic antigen exposure as is the case in HIV infection. PD1up-regulation may also be associated with increased apoptosis in thesesame sets of cells during chronic viral infection (see Petrovas et al,(2009) J Immunol. 183(2):1120-32). PD1 may also play a role intumor-specific escape from immune surveillance. It has been demonstratedthat PD1 is highly expressed in tumor-specific cytotoxic T lymphocytes(CTLs) in both chronic myelogenous leukemia (CIVIL) and acutemyelogenous leukemia (AML). PD1 is also up-regulated in melanomainfiltrating T lymphocytes (TILs) (see Dotti (2009) Blood 114 (8):1457-58). Tumors have been found to express the PD1 ligand PD-L1 or,more rarely, the PD1 ligand PDL2 which, when combined with theup-regulation of PD1 in CTLs, may be a contributory factor in the lossin T-cell functionality and the inability of CTLs to mediate aneffective anti-tumor response. Researchers have shown that in micechronically infected with lymphocytic choriomeningitis virus (LCMV),administration of anti-PD1 antibodies blocked PD1-PDL interaction andwas able to restore some T-cell functionality (proliferation andcytokine secretion), leading to a decrease in viral load (Barber et al(2006) Nature 439(9): 682-687). Additionally, a fully human PD-1specific IgG4 monoclonal antibody has been tested in the clinic in anoncology setting on patients with a variety of disease backgrounds(advanced melanoma, renal cell carcinoma, non-small cell lung cancer,colorectal cancer or prostate cancer). Clinical activity was observed inmelanoma, renal cell and non-small cell lung cancer patients andpreliminary data suggested that detection of PD1 ligand expression bythe tumor prior to treatment correlated with clinical outcome (see Wolfe(2012) Oncology Business Review, July; and Pardoll, ibid).

Another modulator of T-cell activity is the CTLA-4 receptor, and it isalso considered an immunological checkpoint gene. Similar to the T-cellreceptor co-stimulator CD28, CTLA-4 interacts with the CD80 and CD86ligands on antigen presenting cells. But while interaction of theseantigens with CD28 causes activation of T-cells, interaction of CD80 orCD86 with CTLA-4 antagonizes T-cell activation by interfering with IL-2secretion and IL-2 receptor expression, and by inhibiting the expressionof critical cell cycle components. CTLA-4 is not found on the surface ofmost resting T-cells, but is up-regulated transiently after T-cellactivation. Thus, CTLA-4 is also involved in the balance of activatingand inhibiting T-cell activity (see Attia et al. (2005) J Clin Oncol.23(25): 6043-6053). Initial clinical studies involving the use of CTLA 4antibodies in subjects with metastatic melanoma found regression of thedisease (Attia, ibid), but later studies found that subject treated withthe antibodies exhibited side effects of the therapy (immune-relatedadverse events: rashes, colitis, hepatitis etc.) that seemed to berelated to a breaking of self-tolerance. Analysis of this data suggestedthat greater tumor regression as a result of the anti-CTLA4 antibodycorrelated directly with a greater severity of immune-related adverseevents (Weber (2007) Oncologist 12(7): 864-872).

Chimeric Antigen Receptors (CARs) are molecules designed to targetimmune cells to specific molecular targets expressed on cell surfaces.In their most basic form, they are receptors introduced to a cell thatcouple a specificity domain expressed on the outside of the cell tosignaling pathways on the inside of the cell such that when thespecificity domain interacts with its target, the cell becomesactivated. Often CARs are made from variants of T-cell receptors (TCRs)where a specificity domain such as a scFv or some type of receptor isfused to the signaling domain of a TCR. These constructs are thenintroduced into a T-cell allowing the T-cell to become activated in thepresence of a cell expressing the target antigen, resulting in theattack on the targeted cell by the activated T-cell in a non-MHCdependent manner (see Chicaybam et al (2011) Int Rev Immunol30:294-311). Currently, tumor specific CARs targeting a variety of tumorantigens are being tested in the clinic for treatment of a variety ofdifferent cancers. Examples of these cancers and their antigens that arebeing targeted includes follicular lymphoma (CD20 or GD2), neuroblastoma(CD171), non-Hodgkin lymphoma (CD20), lymphoma (CD19), glioblastoma(IL13Rα2), chronic lymphocytic leukemia or CLL and acute lymphocyticleukemia or ALL (both CD19). Virus specific CARs have also beendeveloped to attack cells harboring virus such as HIV. For example, aclinical trial was initiated using a CAR specific for Gp100 fortreatment of HIV (Chicaybam, ibid).

Adoptive cell therapy (ACT) is a developing form of cancer therapy basedon delivering tumor-specific immune cells to a patient in order for thedelivered cells to attack and clear the patient's cancer. ACT ofteninvolves the use of tumor-infiltrating lymphocytes (TILs) which areT-cells that are isolated from a patient's own tumor masses and expandedex vivo to re-infuse back into the patient. This approach has beenpromising in treating metastatic melanoma, where in one study, a longterm response rate of >50% was observed (see for example, Rosenberg etal (2011) Clin Canc Res 17(13): 4550). TILs are a promising source ofcells because they are a mixed set of the patient's own cells that haveT-cell receptors (TCRs) specific for the Tumor associated antigens(TAAs) present on the tumor (Wu et al (2012) Cancer J 18(2):160).However, as stated above, TILs often are up-regulated for PD1expression, presumably due to PDL expression in the tumors, resulting ina population of cells that can target a specific cancer cell andinfiltrate a tumor, but then are unable to kill the cancerous cells. Invitro studies have shown a significant increase in TIL proliferation inresponse to their cognate tumor antigen in the presence of anti-PD1antibodies as compared to stimulation in the absence of the anti-PD1antibody (Wu et al, ibid).).

As useful as it is to develop a technology that will cause a T-cell tore-direct its attention to specific cells such as cancer cells, thereremains the issue that these target cells often express PD-1 ligand. Assuch, the PD1-PD-L1/PD-L2 interaction enables the tumor to escape actionby the CAR-targeted T-cell by deactivating the T-cells and increasingapoptosis and cell exhaustion. Additionally, the PD1-PDL interactionsare also involved in the repression of the T-cell response to HIV, whereincreased expression of both PD1 and PDL leads to T-cell exhaustion.Induction of CTLA-4 expression on activated T-cells is also one of thefirst steps to damping the immune response, and thus a T-cell armed witha CAR might become inactive due to the engagement of this systemdesigned to balance T-cell activation with T-cell inhibition.

Thus, there remains a need for PD1-targeted and/or CTLA-4 modulators,for example PD1 and/or CTLA-4-targeted nucleases or transcriptionrepressors that can be used in research and therapeutic applications.

SUMMARY

The present disclosure relates to development of immunologicalcheckpoint targeted nucleases, for example engineered meganucleases,CRISPR/Cas nuclease systems, zinc finger nucleases (ZFNs) andTALE-nucleases (TALENs) for inactivation of PD1 and/or CTLA-4,optionally in combination with engineered chimeric antigen receptors(CARs) and/or engineered T-cell receptors (TCRs), to prevent or reduceT-cell inhibition. This disclosure also relates to the development oftranscription repressors, for example CRISPR/Cas-, zinc finger- andTALE-based fusion proteins for inactivation of PD1 and/or CTLA-4,optionally in combination with engineered chimeric antigen receptors(CARs) and/or engineered T-cell receptors (TCRs), to prevent or reduceT-cell inhibition.

The present disclosure provides zinc finger proteins specific for humanand rodent PD1 and fusion proteins, including zinc finger proteintranscription factors (ZFP-TFs) or zinc finger nucleases (ZFNs),comprising these PD1-specific zinc finger proteins. The disclosure alsoprovides zinc finger proteins specific for human CTLA-4 and fusionproteins, including zinc finger nucleases (ZFNs), comprising theseCTLA-4-specific zinc finger proteins. The disclosure also providesactive TALE proteins specific for human PD1 and fusion proteins,including TALE nucleases (TALENs) comprising these PD1-specific TALE DNAbinding domains. In certain embodiments, the zinc finger proteincomprising five zinc finger recognition regions ordered from F1 to F5from N-terminus to C-terminus, and wherein the recognition regionscomprise the following amino acid sequences shown in a single row ofTable 2a or Table 2c. In other embodiments, the TAL-effector domain(TALE) comprises a plurality of TALE repeat units, each repeat unitcomprising an Repeat Variable Diresidue (RVD) region that binds to anucleic acid in a target sequence, wherein the TALE binds to a targetsequence as shown SEQ ID NO:29-34 (as shown in Table 5).

The proteins comprising PD1 and/or CTLA-4 specific zinc finger,CRISPR/Cas or TALE proteins of the invention may be used for researchand therapeutic purposes, including for treatment of any disease ordisorder in which PD1 is expressed (e.g., overexpressed), resulting ininactivation or depletion of activated T-cells due to overexpression ofa PDL by a targeted cell and/or a disease or disorder in whichprevention of CTLA-4 engagement will be beneficial. For example, a zincfinger, TALE and/or CRISPR/Cas nuclease targeting of the PD1 locus inT-cells can be used to block PD1-dependent immune suppression in bothchronic infectious diseases and malignancies. Similarly, zinc finger,CRISPR/Cas TALE nuclease and/or targeting of CTLA-4 in T-cells can beused to prevent CTLA-4 mediated T-cell inhibition, for example in thetreatment of cancer. Fusion proteins derived from a linkage of a TALEDNA binding domain and a meganuclease can also be directed to PD1 and/orCTLA-4 for a similar gene knock down or knock out. Transcriptionalrepressor proteins, derived from engineered zinc finger proteins, TALEsand CRISPR/Cas fused to transcription repressor domains can also be usedto prevent PD1 or CTLA-4 mediated T-cell inhibition.

In another aspect of the invention, the fusion proteins comprise zincfinger (ZFN), CRISPR/Cas or TALE (TALEN) nucleases that are specific forthe human PD1 or CTLA-4 genes. In certain embodiments, the zinc fingerdomains of the nuclease fusion proteins specific for PD-1 comprise thenon-naturally occurring recognition helices and/or bind to the targetsites disclosed in U.S. Patent Publication No. 20110136895 (see Tables2a and 2b) and the TALE proteins bind to target sites in PD1 as shown inTables 5a and 5b. In other embodiments, the zinc finger domains of thefusion proteins specific for CTLA-4 comprise the non-naturally occurringrecognition helices shown in Table 2c and/or bind to the target sitesshown in Table 3.

In another aspect, described herein is a CRISPR/Cas system that binds totarget site in a region of interest in a PD1 or CTLA-4 gene in a genome,wherein the CRISPR/Cas system comprises a CRIPSR/Cas nuclease and anengineered crRNA/tracrRNA (or single guide RNA). See, also, U.S. PatentPublication No. 20150056705.

In another aspect, a polynucleotide encoding a nuclease as describedherein is provided, for example a polynucleotides encoding one or morezinc finger nucleases (ZFNs), one or more TALENs, one or moremeganucleases and/or one or more CRISPR/Case nucleases. Thepolynucleotide can comprise DNA, RNA or combinations thereof. In certainembodiments, the polynucleotide comprises a plasmid. In otherembodiments, the polynucleotide encoding the nuclease comprises mRNA.

In one aspect, the methods and compositions of the invention compriseengineered (genetically modified) T-cells. T-cells include, but are notlimited to, helper T-cells (e.g., CD4+ cells), cytotoxic T-cells (e.g.,CD8+), memory T-cells, regulatory T-cells, tumor infiltratinglymphocytes (TILs, CD3+) and the like. In certain embodiments, theT-cells comprise a PD-1 specific nuclease (e.g., for inactivation of PD1in the cell), while in further embodiments, the T-cells comprise a PD-1specific nuclease and at least one transgene donor. In certainembodiments, the T-cells comprise a CTLA-4 specific nuclease (e.g., forinactivation of CTLA-4 in the cell), while in further embodiments, theT-cells comprise a CTLA-4 specific nuclease and at least one transgenedonor. In other embodiments, the genetically modified T-cells aremodified by a nuclease at both an endogenous PD1 gene and endogenousCTLA-4 gene. In still further embodiments, the genetically modifiedT-cells are modified by at least one nuclease at the endogenous TCR, andthe endogenous PD1 and CTLA-4 genes. In some embodiments, the at leastone transgene donor encodes a chimeric antibody receptor (CAR). Incertain embodiments, the CAR donor is integrated into an endogenous PD1and/or CTLA-4 gene. In some embodiments, the CAR donor is integrated bytargeted integration into a safe harbor location. In other embodiments,the CAR-encoding exogenous sequence is introduced via random integrationusing a lentiviral delivery system. In other embodiments, the CAR donoris introduced via random integration using a transposon based deliverysystem.

In other embodiments, the T-cells comprise at least two transgenedonors. In some embodiments, the at least two transgene donors encodesubunits of a T-cell receptor (TCR), e.g. TRAC and TRBC (see UnitedStated Patent Publication No. 20110158957, incorporated by referenceherein). In some instances, the TCR subunits, when expressed from thedonors, comprise a TCR with specificity for a TAA. Some embodimentsinclude engineered TCR chains designed to minimize association with anendogenous TCR. In other embodiments, the endogenous TCR is renderednon-functional via engineered nuclease mediated gene disruption.

In some embodiments, the transgene donor is inserted into the PD-1and/or CTLA-4 locus, such that the transgene is expressed and PD1 orCTLA 4 expression is disrupted. In other embodiments, the engineeredT-cells comprise the PD1 or CTLA-4 specific nuclease, a second nucleasespecific for a safe harbor, and a transgene such that the transgene isinserted into a safe harbor locus (e.g. AAVS1, CCR5 or HPRT) by targetedintegration. See, e.g., U.S. Pat. No. 7,951,925 and U.S. PublicationNos. 20080159996; 201000218264; 20130177983; 20130177960; 20130137104;and 20130122591. In other embodiments, the T-cells comprise a PD1 and/orCTLA-4 specific nuclease, a transgene encoding a CAR, and a secondtransgene encoding another open reading frame. In some embodiments, thesecond transgene encodes a suicide gene. In some embodiments, theT-cells comprise a PD1 and/or CTLA-4 specific nuclease, a transgeneencoding a CAR and a set of transgenes encoding a TAA-specific TCR. Inother aspects, the donor transgenes are integrated into the T-cellsprior to the nucleases being integrated, while in some aspects, both thedonor transgenes and the nucleases are introduced into the T-celltogether.

In another aspect, described herein are methods of modifying a T-cell.In certain embodiments, PD1 and/or CTLA-4 expression in the T-cell isreduced or inactivated, for example using a zinc finger or TALEtranscription factor, a zinc finger nuclease and/or a TALEN and/orCRIPSR/Cas system. In certain embodiments, the methods further compriseintroducing one or more exogenous sequences (e.g., transgenes) into anyof the PD1- and/or CTLA-4 modified cells as described herein, forexample a transgene encoding a CAR and/or a set of transgenes encoding aTAA-specific TCR. In certain embodiments, the T-cell is activated, forexample bead-activation as described in U.S. Publication No.20080311095. In other embodiments, the T-cells are resting. In someaspects, the T-cell is a TIL. In other aspects, the T-cell comprises aT-cell line derived from a TIL. In some embodiments, the TIL ischaracterized for its HLA subtypes, and in other embodiments, the TILscarry specifically engineered HLA knock-outs and/or knock-ins (see USPatent Publication No. 20120060230, incorporated by reference herein).

In another aspect, the methods of the invention comprise a compositionfor therapeutic treatment of a subject in need thereof. In someembodiments, the composition comprises engineered T-cells or TILscomprising a PD1 or CTLA-4 specific nuclease, a safe harbor specificnuclease, at least one transgene donor encoding a CAR, a secondtransgene donor and any combinations of nucleases and donors thereof. Insome aspects, the transgene donors encode a TAA-specific TCR. In otherembodiments, the compositions comprise engineered T-cells or TILscomprising a PD1 or CTLA-4 specific nuclease and a transgene donorencoding a CAR.

In another aspect, provided herein are methods and compositions for theregulation of the PD1 or CTLA-4 gene. In certain embodiments, themethods comprise introducing a nuclease (e.g., ZFN, TALEN, CRISPR/Cas,meganuclease, TALE-meganuclease fusions, etc.) that binds to andmodifies a PD1 or CTLA-4 gene. In certain embodiments, the nuclease is afusion protein comprising a zinc finger or TALE fusion protein that isengineered to bind to a target site at the PD1 or CTLA-4 locus (orpolynucleotide encoding the fusion protein) into cells from a subjectwith a disease or disorder to prevent or treat the disease or disorder.In some embodiments, the methods comprise introducing a transcriptionregulator (e.g., ZFP-TF, TALE-TF, CRIPSR/Cas-TF etc.) into a cell thatbinds to and represses expression of a PD1 or CTLA-4 gene. In someembodiments, the disease or disorder is a cancer or a malignancy, andthe zinc finger or TALE fusion protein is a nuclease or a fusion proteincomprising a transcription repression domain. In other embodiments, thenuclease comprises a CRISPR/Cas nuclease system. Non-limiting examplesof cancers that can be treated and/or prevented include lung carcinomas,pancreatic cancers, liver cancers, melanomas, bone cancers, breastcancers, colorectal cancers, leukemias, ovarian cancers, lymphomas,brain cancers and the like.

A kit, comprising the ZFNs, TALENs and/or CRIPSR/Cas system of theinvention, is also provided. The kit may comprise nucleic acids encodingthe ZFNs, TALENs or CRISPR/Cas system, (e.g. RNA molecules or ZFP, TALENor Cas9 encoding genes contained in a suitable expression vector) andengineered sg RNA if needed, or aliquots of the nuclease proteins, donormolecules, suitable host cell lines, instructions for performing themethods of the invention, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a gel showing the activity (as measured by % indeldetection) of PD1-specific TALENs in K562 cells using the Cel-I assay(described in the text). Lane designations are as in text and % indelsdetected is indicated at the bottom of each lane.

FIG. 2 is a graph depicting percent non-homologous end joining events(NHEJ) as determined by Cel-1 assay) using the indicated nucleases andconditions. “R5” refers to cells electroporated with CCR5-specific ZFNmRNA (see, e.g., U.S. Pat. No. 7,951,925). “PD1” refers to cellselectroporated with PD1-specific ZFN mRNA (see, e.g., U.S. PublicationNo. 20110136895). “C1” and “C3” refer to electroporation conditions. Theleft bar of each pair shows the % NHEJ at the indicated conditions 3days after mRNA electroporation (EPD3) and the right bar shows % NHEJ atthe indicated conditions 5 days after mRNA electroporation (EPD5).

DETAILED DESCRIPTION

Described herein are compositions and methods for modulation of PD1and/or CTLA-4. These compositions and methods are useful for researchand therapeutic applications and involve the use of genome editing viaengineered nucleases to disrupt the PD1 and/or CTLA-4 gene. Theinventive methods also include PD1 or CTLA-4 specific zinc finger orTALE DNA binding domain fused to transcription repressors to preventexpression of the PD1 or CTLA-4 genes. The methods and compositionsincluded also describe the use of chimeric antigen receptors foractivation of T-cells against specific cell targets in T-cells with aPD1 and/or CTLA-4 disruption.

Interaction of PD1, expressed on a T-cell, with PD1-ligand can causedeactivation of the T-cell. Some cancer cells express PD1 ligands, andin this way, are able to avoid immune surveillance and are able toproliferate despite the presence of T-cells that are capable, in theabsence of PD-1 ligand, of destroying that cancer cell. Furthermore,even if the T-cell has been modified such that it expresses a CAR thatactivates and redirects that T-cell to a cell bearing a particularmarker, expression of PD1 ligands by that targeted cell can causedesensitization of the activated T-cell, and the desensitized T-cellwill then no longer act on the targeted cell.

CTLA-4 expression is induced upon T-cell activation on activatedT-cells, and competes for binding with the antigen presenting cellactivating antigens CD80 and CD86. Interaction of CTLA-4 with CD80 orCD86 causes T-cell inhibition and serves to maintain balance of theimmune response. However, inhibition of the CTLA-4 interaction with CD80or CD86 may prolong T-cell activation and thus increase the level ofimmune response to a cancer antigen. The present invention describesinhibition of the CTAL-4 interaction via a blockade of its expressionwith a zinc finger or TALE-transcription factor fusion, or via treatmentof the T-cell with a CTLA-4 specific nuclease to knock out the gene.

CAR technology offers the potential for designer T-cells that willattack specific cells, where the target of those T-cells is chosen bythe investigator. Medical researchers have long suggested that T-cellsregularly remove malignant or aberrant cells as a matter of course, andyet there are some cancers that are able to escape, perhaps through theuse of PD-1 ligand driven immune response damping. Thus, as promising asthe use of CARs appears to be, the combination of T-cells engineered toexpress CARs (which targets them to particular tumor cells) incombination with transcription factors and/or nucleases (e.g., zincfinger, TALE, and/or CRISPR/Cas based) to repress or knock out PD1 orCTLA-4 expression in those same cells provides novel cell and animalmodels for, and methods of, researching and treating various diseasesand disorders.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. Each TALErepeat unit includes 1 or 2 DNA-binding residues making up the RepeatVariable Diresidue (RVD), typically at positions 12 and/or 13 of therepeat. The natural (canonical) code for DNA recognition of these TALEshas been determined such that an HD sequence at positions 12 and 13leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds toG or A, and NG binds to T and non-canonical (atypical) RVDs are alsoknown. See, U.S. Patent Publication No. 20110301073, incorporated byreference herein in its entirety.

A “CRISPR/Cas nuclease” or “CRISPR/Cas nuclease system” includes anon-coding RNA molecule (guide) RNA that binds to DNA and Cas proteins(Cas9) with nuclease functionality (e.g., two nuclease domains). See,e.g., U.S. Patent Publication No. 20150056705.

In any of the methods described herein, additional pairs of zinc-fingerand/or TALEN proteins can be used for additional double-strandedcleavage of additional target sites within the cell. In addition, aCRISPR/Cas system may be used alone or in combination with ZFNs and/orTALENs to induce additional double strand breaks.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No.20110301073.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988;U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO01/88197; WO 02/099084 and U.S. Publication No. 20110301073.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;”“+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 20050064474, 20070218528, 20080131962 and20110201055, incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value there between or there above), preferablybetween about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides inlength. “Chromatin” is the nucleoprotein structure comprising thecellular genome. Cellular chromatin comprises nucleic acid, primarilyDNA, and protein, including histones and non-histone chromosomalproteins. The majority of eukaryotic cellular chromatin exists in theform of nucleosomes, wherein a nucleosome core comprises approximately150 base pairs of DNA associated with an octamer comprising two each ofhistones H2A, H2B, H3 and H4; and linker DNA (of variable lengthdepending on the organism) extends between nucleosome cores. A moleculeof histone H1 is generally associated with the linker DNA. For thepurposes of the present disclosure, the term “chromatin” is meant toencompass all types of cellular nucleoprotein, both prokaryotic andeukaryotic. Cellular chromatin includes both chromosomal and episomalchromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

A “chronic infectious disease” is a disease caused by an infectiousagent wherein the infection has persisted. Such a disease may includehepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HSV-6, HSV-II,CMV, and EBV), and HIV/AIDS. Non-viral examples may include chronicfungal diseases such Aspergillosis, Candidiasis, Coccidioidomycosis, anddiseases associated with Cryptococcus and Histoplasmosis. None limitingexamples of chronic bacterial infectious agents may be Chlamydiapneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis.

The term “autoimmune disease” refers to any disease or disorder in whichthe subject mounts a destructive immune response against its owntissues. Autoimmune disorders can affect almost every organ system inthe subject (e.g., human), including, but not limited to, diseases ofthe nervous, gastrointestinal, and endocrine systems, as well as skinand other connective tissues, eyes, blood and blood vessels. Examples ofautoimmune diseases include, but are not limited to Hashimoto'sthyroiditis, Systemic lupus erythematosus, Sjogren's syndrome, Graves'disease, Scleroderma, Rheumatoid arthritis, Multiple sclerosis,Myasthenia gravis and Diabetes.

The term “cancer” as used herein is defined as a hyperproliferation ofcells whose unique trait—loss of normal controls—results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis.With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer,breast cancer, cancer of the anus, anal canal, or anorectum, cancer ofthe eye, cancer of the intrahepatic bile duct, cancer of the joints,cancer of the neck, gallbladder, or pleura, cancer of the nose, nasalcavity, or middle ear, cancer of the oral cavity, cancer of the vulva,chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer,esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinalcarcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer,lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiplemyeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, solid tumors, stomach cancer,testicular cancer, thyroid cancer, ureter cancer, and urinary bladdercancer. As used herein, the term “tumor” refers to an abnormal growth ofcells or tissues of the malignant type, unless otherwise specificallyindicated and does not include a benign type tissue. The term “inhibitsor inhibiting” as used herein means reducing growth/replication.

The term “immunological checkpoint gene” refers to any gene that isinvolved in an inhibitory process (e.g., feedback loop) that acts toregulate the amplitude of an immune response, for example an immuneinhibitory feedback loop that mitigates uncontrolled propagation ofharmful immune responses. These responses include contributing to amolecular shield that protects against collateral tissue damage thatmight occur during immune responses to infections and/or maintenance ofperipheral self-tolerance. Non-limiting examples of immunologicalcheckpoint genes include members of the extended CD28 family ofreceptors and their ligands as well as genes involved in co-inhibitorypathways (e.g., CTLA-4 and PD-1).

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain); fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra) andfusions between nucleic acids and proteins (e.g., CRISPR/Cas nucleasesystem). Examples of the second type of fusion molecule include, but arenot limited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion molecule in a cell can result from delivery ofthe fusion molecule to the cell, for instance for fusion proteins bydelivery of the fusion protein to the cell or by delivery of apolynucleotide encoding the fusion protein to a cell, wherein thepolynucleotide is transcribed, and the transcript is translated, togenerate the fusion protein. Trans-splicing, polypeptide cleavage andpolypeptide ligation can also be involved in expression of a protein ina cell. Methods for polynucleotide and/or polypeptide delivery to cellsare presented elsewhere in this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the expressionlevel of a gene. Modulation of expression can include, but is notlimited to, gene activation and gene repression. Modulation may also becomplete, i.e. wherein gene expression is totally inactivated or isactivated to wildtype levels or beyond; or it may be partial, whereingene expression is partially reduced, or partially activated to somefraction of wildtype levels. “Eukaryotic” cells include, but are notlimited to, fungal cells (such as yeast), plant cells, animal cells,mammalian cells and human cells (e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP, TALEor CasDNA-binding domain is fused to a cleavage domain, the DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the DNA-binding domain portion is able to bind itstarget site and/or its binding site, while the cleavage domain is ableto cleave DNA in the vicinity of the target site. Similarly, withrespect to a fusion polypeptide in which a ZFP, TALE or CasDNA-bindingdomain is fused to an activation or repression domain, the DNA-bindingdomain and the activation or repression domain are in operative linkageif, in the fusion polypeptide, the DNA-binding domain portion is able tobind its target site and/or its binding site, while the activationdomain is able to upregulate gene expression or the repression domain isable to downregulate gene expression. ZFPs fused to domains capable ofregulating gene expression are collectively referred to as “ZFP-TFs” or“zinc finger transcription factors”, while TALEs fused to domainscapable of regulating gene expression are collectively referred to as“TALE-TFs” or “TALE transcription factors” and CRISPR/Cas proteinslinked to domains capable of regulating gene expression are collectivelyreferred to “CRISPR/Cas TFs”.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

A “chimeric antigen receptor” (CAR) is an artificially constructedhybrid protein or polypeptide comprising a specificity or recognition(i.e. binding) domain linked to an immune receptor responsible forsignal transduction in lymphocytes. Most commonly, the binding domain isderived from a Fab antibody fragment that has been fashioned into asingle chain scFv via the introduction of a flexible linker between theantibody chains within the specificity domain. Other possiblespecificity domains can include the signaling portions of hormone orcytokine molecules, the extracellular domains of receptors, and peptideligands or peptides isolated by library (e.g. phage) screening (seeRamos and Dotti, (2011) Expert Opin Bio Ther 11(7): 855). Flexibilitybetween the signaling and the binding portions of the CAR may be adesirable characteristic to allow for more optimum interaction betweenthe target and the binding domain, so often a hinge region is included.One example of a structure that can be used is the CH2-CH3 region froman immunoglobulin such as an IgG molecule. The signaling domain of thetypical CAR comprises intracellular domains of the TCR-CD3 complex suchas the zeta chain. Alternatively, the γ chain of an Fc receptor may beused. The transmembrane portion of the typical CAR can comprisetransmembrane portions of proteins such as CD4, CD8 or CD28 (Ramos andDotti, ibid). Characteristics of some CARs include their ability toredirect T-cell specificity and reactivity toward a selected target in anon-MHC-restricted manner. The non-MHC-restricted target recognitiongives T-cells expressing CARs the ability to recognize a targetindependent of antigen processing, thus bypassing a major mechanism oftumor escape.

So called “first generation” CARs often comprise a single internalsignaling domain such as the CD3 zeta chain, and are thought to besomewhat ineffectual in the clinic, perhaps due to incompleteactivation. To increase performance of T-cells bearing these CARs,second generation CARs have been generated with the ability of provingthe T-cell additional activation signals by including anotherstimulatory domain, often derived from the intercellular domains ofother receptors such as CD28, CD134/OX40, CD137/4-1BB, Lck, ICOS andDAP10. Additionally, third generation CARs have also been developedwherein the CAR contains three or more stimulatory domains (Ramos andDotti, ibid).

In some instances, CAR can comprise an extracellular hinge domain,transmembrane domain, and optionally, an intracellular hinge domaincomprising CD8 and an intracellular T-cell receptor signaling domaincomprising CD28, 4-1BB, and CD3.zeta. CD28 is a T-cell marker importantin T-cell co-stimulation. CD8 is also a T-cell marker. 4-1BB transmits apotent costimulatory signal to T-cells, promoting differentiation andenhancing long-term survival of T lymphocytes. CD3.zeta. associates withTCRs to produce a signal and contains immunoreceptor tyrosine-basedactivation motifs (ITAMs). In other instances, CARs can comprise anextracellular hinge domain, transmembrane domain, and intracellularT-cell signaling domain comprising CD28 and CD3.zeta. In furtherinstances, CARs can comprise an extracellular hinge domain andtransmembrane domain comprising CD8 and an intracellular T-cell receptorsignaling domain comprising CD28 and CD3.zeta.

Overview

Described herein are DNA-binding molecules (e.g., zinc finger, TALEand/or CRISPR/Cas nucleases and/or transcription factors targeted to thePD1 gene and/or CTLA-4 gene as well as compositions comprising andmethods of using these nucleases and artificial transcription factorsfor treatment of disease or disorders, particularly disorders in whichPD1 or PD1 ligands are undesirably expressed on cells of the immunesystem, cancers and/or autoimmune diseases and/or diseases or disordersin which repression of CTLA-4 expression would be beneficial. Fortreatment of a subject with a disease or disorder that is ameliorated bythe modulation of the PD1/PD1 ligand interaction, or CTLA-4 mediatedT-cell inhibition, the nucleases described herein can be introduced invivo or ex vivo into cells (e.g., primary cells such as T-cells isolatedfrom a subject afflicted with such a disease) to prevent expression ofPD1 or CTLA-4 on the treated cells. Following nuclease treatment, thePD1 or CTLA-4 knock out T-cells may be reintroduced into the subject foruse as a medicament in the treatment of a chronic infectious disease orcancer, or maybe be expanded prior to re-introduction. Alternatively,modulation of the PD1 or CTLA-4 loci may occur in vivo throughintroduction of the necessary nucleases or engineered transcriptionfactors into a subject. Similarly, stem cells may be used that have beentreated with the PD1- and/or CTLA-4 specific nucleases (e.g., ZFNs,CRISPR/Cas nuclease systems and/or TALENs). These cells can be infusedinto an afflicted subject for treatment of such a medical condition.

In some instances, the PD1 or CTLA-4 specific nucleases or transcriptionfactors may be used in concert with chimeric antigen receptors. Thus,the invention contemplates, for example, methods in which a CAR thatspecifically targets a protein or non-protein tumor antigen isintroduced into a T-cell such that the T-cell bearing such a CAR willbecome activated in the presence of the antigen. The use of a CAR in acell that has also been, or will be, treated with PD1- and/orCTLA-4-specific nucleases or transcription factors, in which the PD1 orCTLA-4 gene(s) is(are) knocked out or otherwise similarly modulated,results in a T-cell expressing a CAR of interest that is resistant tothe PD1 ligand produced by the cancer cell and thus is not subject toPD-1 mediated T-cell exhaustion and/or resistant to CTLA-4 mediatedT-cell inhibition.

Numerous cancer antigens are known in the art and may be targeted byspecific CARs. By way of non-limiting examples, see Table 1 for tumorassociated antigens that may be targeted by CARs (see Ramos and Dotti,ibid, and Orentas et al (2012), Front in Oncol 2:1).

TABLE 1 Tumor associated antigens suitable for CAR targeting Tumor typeAntigen Description Gastrointenstinal EGP2/EpCam Epithelial glycoprotein2/Epithelial cell adhesion molecule Gastrointenstinal EGP40 Epithelialglycoprotein 40 Gastrointenstinal TAG72/CA72-4 Tumor associatedglycoprotein 72/cancer antigen 72-4 Glioblastoma IL13Rα2 Interleukin 13receptor alpha-2 subunit Kidney G250/MN/CA IX Carbonic anhydrase IXLymphoid malignancies CD19 Lymphoid malignancies CD52 Lymphoidmalignancies CD33 Lymphoid malignancies CD20 Membrane-spanning 4-domainssubfamily A member 1 Lymphoid malignancies TSLPR (CRLF2) Lymphoidmalignancies CD22 Sialic acid-binding Ig-like lectin 2 Lymphoidmalignancies CD30 TNF receptor superfamily member 8 Lymphoidmalignancies κ Kappa light chain Melanoma GD3 GD3-Ganglioside MelanomaHLA-A1 + MAGE-1 Human leukocyte antigen A1 + Melanoma antigen 1Neuroblastoma/Neural CD171 L1 cell adhesion molecule tumorsNeuroblastoma/Neural ALK Anaplastic lymphoma kinase tumorsNeuroblastoma/Neural GD2 GD2-Ganglioside tumors Neuroblastoma/NeuralCD47 tumors Neuroblastoma/Neural EGFRvIII tumors Neuroblastoma/NeuralNCAM Neural cell adhesion molecule tumors Ovary FBP/αFR Folate bindingprotein/alpha folate receptor Ovary Le(Y) Lewis-Y antigen Ovary MUC1Mucin 1 Prostate PSCA Prostate stem cell antigen Prostate PSMAProstate-specific membrane antigen Rhadbomyosarcoma FGFR4 Fibroblastgrowth factor receptor 4 Rhadbomyosarcoma FAR Fetal acetylcholinereceptor Several solid tumors CEA Carcinoembryonic antigen Several solidtumors ERBB2/HER2 Avian ertyroblastic leukemia viral oncogene homolog2/Human epidermal growth factor receptor 2 Several solid tumors ERBB3 +ERBB4 Avian erthroblastic leukemia viral oncogene homology 3 + 4 Severalsolid tumors Mesothelin Various tumors CD44v6 Hyaluronate receptorvariant 6 Various tumors B7-H3 Adhesion receptor Various tumorsGlypican-3,5 Cell surface peptidoglycan Various tumors ROR1 Varioustumors Survivin Anti-apoptotic molecule Various tumors FOLR1 α folatereceptor Various tumors WT1 Wilm's tumor antigen Various tumors CD70Various tumors VEGFR2/FLK/KDR Vascular endothelial growth factor 2/Fetalliver kinase 1/Kinase domain insert

Further, recombinant expression vectors, for example vectors including asuicide gene, or such gene may be introduced separately. As used herein,the term “suicide gene” refers to a gene that causes the cell expressingthe suicide gene to die. The suicide gene can be a gene that conferssensitivity to an agent, e.g., a drug that acts upon the cell in whichthe gene is expressed, and causes the cell to die when the cell iscontacted with or exposed to the agent. Suicide genes are known in theart (see, for example, Suicide Gene Therapy: Methods and Reviews,Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeuticsat the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press,2004) and include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase,and nitroreductase.

DNA-Binding Domains

Described herein are compositions comprising a DNA-binding domain thatspecifically binds to a target site in a PD1 or CTLA-4 locus. AnyDNA-binding domain can be used in the compositions and methods disclosedherein, including but not limited to a zinc finger DNA-binding domain, aTALE DNA binding domain, CRISPR/Cas DNA-binding nuclease system or aDNA-binding domain from a meganuclease.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein or TALE DNA-binding protein. Preferably, the zinc finger proteinis non-naturally occurring in that it is engineered to bind to a targetsite of choice. See, for example, Beerli et al. (2002) NatureBiotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal etal. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr.Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261;6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317;7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent PublicationNos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated hereinby reference in their entireties. In other embodiments, the DNA bindingdomain comprises a TALE DNA binding domain (see, co-owned US Patentpublication No. 20110301073, incorporated by reference in its entiretyherein).

An engineered zinc finger or TALE DNA binding domain can have a novelbinding specificity, compared to a naturally-occurring zinc finger orTALE protein. Engineering methods include, but are not limited to,rational design and various types of selection. Rational designincludes, for example, using databases comprising triplet (orquadruplet) nucleotide sequences and individual zinc finger amino acidsequences, in which each triplet or quadruplet nucleotide sequence isassociated with one or more amino acid sequences of zinc fingers whichbind the particular triplet or quadruplet sequence. See, for example,co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated byreference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins or TALEs may belinked together using any suitable linker sequences, including forexample, linkers of 5 or more amino acids in length. See, also, U.S.Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linkersequences 6 or more amino acids in length. The proteins described hereinmay include any combination of suitable linkers between the individualzinc fingers of the protein. In addition, enhancement of bindingspecificity for zinc finger binding domains has been described, forexample, in co-owned WO 02/077227.

Selection of target sites; ZFPs or TALEs and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S. Pat.Nos. 6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, DNA-bindingdomains may be linked together using any suitable linker sequences,including for example, linkers of 5 or more amino acids in length. See,also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplarylinker sequences 6 or more amino acids in length. The proteins describedherein may include any combination of suitable linkers between theindividual DNA-binding domains of the protein.

Alternatively, the DNA-binding domain may be derived from a nuclease.For example, the recognition sequences of homing endonucleases andmeganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI,I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue. In addition, theDNA-binding specificity of homing endonucleases and meganucleases can beengineered to bind non-natural target sites. See, for example, Chevalieret al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic AcidsRes. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques etal. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128.

In other embodiments, the DNA-binding domain is in a CRISPR/Cas nucleasesystem, guided by, for example, an RNA molecule.

In certain embodiments, the DNA binding domain is an engineered zincfinger protein that binds (in a sequence-specific manner) to a targetsite in a PD1 or CTLA-4 locus and modulates expression of PD1 or CTLA-4.PD1 and CTLA-4 target sites typically include at least one zinc fingerbut can include a plurality of zinc fingers (e.g., 2, 3, 4, 5, 6 or morefingers). Usually, the ZFPs include at least three fingers. Certain ofthe ZFPs include four, five or six fingers. The ZFPs that include threefingers typically recognize a target site that includes 9 or 10nucleotides; ZFPs that include four fingers typically recognize a targetsite that includes 12 to 14 nucleotides; while ZFPs having six fingerscan recognize target sites that include 18 to 21 nucleotides. The ZFPscan also be fusion proteins that include one or more regulatory domains,wherein these regulatory domains can be transcriptional activation orrepression domains.

In other embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector DNAbinding domain. See, e.g., U.S. Patent Publication No. 20110301073,incorporated by reference in its entirety herein. The plant pathogenicbacteria of the genus Xanthomonas are known to cause many diseases inimportant crop plants. Pathogenicity of Xanthomonas depends on aconserved type III secretion (T3 S) system which injects more than 25different effector proteins into the plant cell. Among these injectedproteins are transcription activator-like effectors (TALE) which mimicplant transcriptional activators and manipulate the plant transcriptome(see Kay et al (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TALEs is AvrBs3 from Xanthomonas campestris pv.Vesicatoria (see Bonas et al (1989) Mol Gen Genet 218: 127-136 andWO2010079430). TALEs contain a centralized domain of tandem repeats,each repeat containing approximately 34 amino acids, which are key tothe DNA binding specificity of these proteins. In addition, they containa nuclear localization sequence and an acidic transcriptional activationdomain (for a review see Schornack S, et al (2006) J Plant Physiol163(3): 256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg1 1 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas.

The CRISPR/Cas System

Compelling evidence has recently emerged for the existence of anRNA-mediated genome defense pathway in archaea and many bacteria thathas been hypothesized to parallel the eukaryotic RNAi pathway (forreviews, see Godde and Bickerton, 2006. J. Mol. Evol. 62: 718-729;Lillestol et al., 2006. Archaea 2: 59-72; Makarova et al., 2006. Biol.Direct 1: 7.; Sorek et al., 2008. Nat. Rev. Microbiol. 6: 181-186).Known as the CRISPR-Cas system or prokaryotic RNAi (pRNAi), the pathwayis proposed to arise from two evolutionarily and often physically linkedgene loci: the CRISPR (clustered regularly interspaced short palindromicrepeats) locus, which encodes RNA components of the system, and the cas(CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002.Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res.30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al.,2005. PLoS Comput. Biol. 1: e60). CRISPR loci in microbial hosts containa combination of CRISPR-associated (Cas) genes as well as non-coding RNAelements capable of programming the specificity of the CRISPR-mediatednucleic acid cleavage. The individual Cas proteins do not sharesignificant sequence similarity with protein components of theeukaryotic RNAi machinery, but have analogous predicted functions (e.g.,RNA binding, nuclease, helicase, etc.) (Makarova et al., 2006. Biol.Direct 1: 7). The CRISPR-associated (cas) genes are often associatedwith CRISPR repeat-spacer arrays. More than forty different Cas proteinfamilies have been described. Of these protein families, Cas1 appears tobe ubiquitous among different CRISPR/Cas systems. Particularcombinations of cas genes and repeat structures have been used to define8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, andMtube), some of which are associated with an additional gene moduleencoding repeat-associated mysterious proteins (RAMPs). More than oneCRISPR subtype may occur in a single genome. The sporadic distributionof the CRISPR/Cas subtypes suggests that the system is subject tohorizontal gene transfer during microbial evolution.

There are three types of CRISPR/Cas systems which all incorporate RNAsand Cas proteins. Types I and III both have Cas endonucleases thatprocess the pre-crRNAs, that, when fully processed into crRNAs, assemblea multi-Cas protein complex that is capable of cleaving nucleic acidsthat are complementary to the crRNA.

The Type II CRISPR (exemplified by Cas9) is one of the most wellcharacterized systems and carries out targeted DNA double-strand breakin four sequential steps. First, two non-coding RNA, the pre-crRNA arrayand tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNAhybridizes to the repeat regions of the pre-crRNA and mediates theprocessing of pre-crRNA into mature crRNAs containing individual spacersequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to thetarget DNA via Watson-Crick base-pairing between the spacer on the crRNAand the protospacer on the target DNA next to the protospacer adjacentmotif (PAM), an additional requirement for target recognition. Finally,Cas9 mediates cleavage of target DNA to create a double-stranded breakwithin the protospacer. Activity of the CRISPR/Cas system comprises ofthree steps: (i) insertion of alien DNA sequences into the CRISPR arrayto prevent future attacks, in a process called ‘adaptation,’ (ii)expression of the relevant proteins, as well as expression andprocessing of the array, followed by (iii) RNA-mediated interferencewith the alien nucleic acid. Thus, in the bacterial cell, several of theso-called ‘Cas’ proteins are involved with the natural function of theCRISPR/Cas system.

The primary products of the CRISPR loci appear to be short RNAs thatcontain the invader targeting sequences, and are termed guide RNAs orprokaryotic silencing RNAs (psiRNAs) based on their hypothesized role inthe pathway (Makarova et al. (2006) Biol. Direct 1:7; Hale et al. (2008)RNA 14: 2572-2579). RNA analysis indicates that CRISPR locus transcriptsare cleaved within the repeat sequences to release ^(˜)60- to 70-nt RNAintermediates that contain individual invader targeting sequences andflanking repeat fragments (Tang et al. (2002) Proc. Natl. Acad. Sci. 99:7536-7541; Tang et al. (2005) Mol. Microbiol. 55:469-481; Lillestol etal. (2006) Archaea 2:59-72; Brouns et al. (2008) Science 321: 960-964;Hale et al. (2008) RNA 14:2572-2579). In the archaeonPyrococcusfuriosus, these intermediate RNAs are further processed toabundant, stable ^(˜)35- to 45-nt mature psiRNAs (Hale et al. (2008) RNA14: 2572-2579).

In type II CRISPR/Cas systems, crRNAs are produced using a differentmechanism where a trans-activating RNA (tracrRNA) complementary torepeat sequences in the pre-crRNA, triggers processing by a doublestrand-specific RNase III in the presence of the Cas9 protein. Cas9 isthen able to cleave a target DNA that is complementary to the maturecrRNA however cleavage by Cas 9 is dependent both upon base-pairingbetween the crRNA and the target DNA, and on the presence of a shortmotif in the crRNA referred to as the PAM sequence (protospacer adjacentmotif) (see Qi et al. (2013) Cell 152:1173). In addition, the tracrRNAmust also be present as it base pairs with the crRNA at its 3′ end, andthis association triggers Cas9 activity.

The requirement of the crRNA-tracrRNA complex can be avoided by use ofan engineered “single-guide RNA” (sgRNA) that comprises the hairpinnormally formed by the annealing of the crRNA and the tracrRNA (see,Jinek et al. (2012) Science 337:816 and Cong et al. (2013)Sciencexpress/10.1126/science.1231143). In S. pyrogenes, the engineeredtracrRNA:crRNA fusion, or the sgRNA, guides Cas9 to cleave the targetDNA when a double strand RNA:DNA heterodimer forms between the Casassociated RNAs and the target DNA. This system comprising the Cas9protein and an engineered sgRNA containing a PAM sequence has been usedfor RNA guided genome editing (see Ramalingam, ibid) and has been usefulfor zebrafish embryo genomic editing in vivo (see Hwang et al. (2013)Nature Biotechnology 31(3):227) with editing efficiencies similar toZFNs and TALENs.

Cas Proteins

The Cas9 protein has at least two nuclease domains: one nuclease domainis similar to a HNH endonuclease, while the other resembles a Ruvendonuclease domain. The HNH-type domain appears to be responsible forcleaving the DNA strand that is complementary to the crRNA while the Ruvdomain cleaves the non-complementary strand.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof.

“Cas polypeptide” encompasses a full-length Cas polypeptide, anenzymatically active fragment of a Cas polypeptide, and enzymaticallyactive derivatives of a Cas polypeptide or fragment thereof. Suitablederivatives of a Cas polypeptide or a fragment thereof include but arenot limited to mutants, fusions, covalent modifications of Cas proteinor a fragment thereof.

Cas proteins and Cas polypeptides may be obtainable from a cell orsynthesized chemically or by a combination of these two procedures. Thecell may be a cell that naturally produces Cas protein, or a cell thatnaturally produces Cas protein and is genetically engineered to producethe endogenous Cas protein at a higher expression level or to produce aCas protein from an exogenously introduced nucleic acid, which nucleicacid encodes a Cas that is same or different from the endogenous Cas. Insome case, the cell does not naturally produce Cas protein and isgenetically engineered to produce a Cas protein.

The CRISPR/Cas system can also be used to inhibit gene expression. Leiet al. (2013) Cell 152(5):1173-1183) have shown that a catalyticallydead Cas9 lacking endonuclease activity, when coexpressed with a guideRNA, generates a DNA recognition complex that can specifically interferewith transcriptional elongation, RNA polymerase binding, ortranscription factor binding. This system, called CRISPR interference(CRISPRi), can efficiently repress expression of targeted genes.

Additionally, Cas proteins have been developed which comprise mutationsin their cleavage domains to render them incapable of inducing a DSB,and instead introduce a nick into the target DNA (“Cas9 nicking enzyme”,see Cong et al., ibid). In particular, the Cas nuclease comprises twonuclease domains, the HNH and RuvC-like, for cleaving the sense and theantisense strands of the target DNA, respectively. The Cas nuclease canthus be engineered such that only one of the nuclease domains isfunctional, thus creating a Cas nickase. See, e.g., Jinek et al., ibid,and Cong et al., ibid.

The Cas proteins of the invention may be mutated to alter functionality.Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237.

RNA Components of CRISPR/Cas

The Cas9 related CRISPR/Cas system comprises two RNA non-codingcomponents: tracrRNA and a pre-crRNA array containing nuclease guidesequences (spacers) interspaced by identical direct repeats (DRs). Touse a CRISPR/Cas system to accomplish genome engineering, both functionsof these RNAs must be present (see Cong et al, (2013) Sciencexpress1/10.1126/science 1231143). In some embodiments, the tracrRNA andpre-crRNAs are supplied via separate expression constructs or asseparate RNAs. In other embodiments, a chimeric RNA is constructed wherean engineered mature crRNA (conferring target specificity) is fused to atracrRNA (supplying interaction with the Cas9) to create a chimericcr-RNA-tracrRNA hybrid (also termed a single guide RNA). (see, Jinek,ibid and Cong, ibid).

Chimeric or sgRNAs can be engineered to comprise a sequencecomplementary to any desired target. The RNAs comprise 22 bases ofcomplementarity to a target and of the form G[n19], followed by aprotospacer-adjacent motif (PAM) of the form NGG. Thus, in one method,sgRNAs can be designed by utilization of a known ZFN target in a gene ofinterest by (i) aligning the recognition sequence of the ZFN heterodimerwith the reference sequence of the relevant genome (human, mouse, or ofa particular plant species); (ii) identifying the spacer region betweenthe ZFN half-sites; (iii) identifying the location of the motif G[N20]GGthat is closest to the spacer region (when more than one such motifoverlaps the spacer, the motif that is centered relative to the spaceris chosen); (iv) using that motif as the core of the sgRNA. This methodadvantageously relies on proven nuclease targets. Alternatively, sgRNAscan be designed to target any region of interest simply by identifying asuitable target sequence that conforms to the G[n20]GG formula.

Target Sites

As described in detail above, DNA domains (ZFPs, TALEs, CRISPR RNAs,meganucleases) can be engineered to bind to any sequence of choice in alocus. An engineered DNA-binding domain can have a novel bindingspecificity, compared to a naturally-occurring DNA-binding domain.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual (e.g., zinc finger) amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of DNA binding domain which bind the particulartriplet or quadruplet sequence. See, for example, co-owned U.S. Pat.Nos. 6,453,242 and 6,534,261, incorporated by reference herein in theirentireties. Rational design of TAL-effector domains can also beperformed. See, e.g., U.S. Publication No. 20110301073.

Exemplary selection methods applicable to DNA-binding domains, includingphage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO00/27878; WO 01/88197 and GB 2,338,237.

Selection of target sites; nucleases and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S.Patent Publication Nos. 20050064474 and 20060188987, incorporated byreference in their entireties herein.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949 for exemplary linker sequences 6 or more aminoacids in length. The proteins described herein may include anycombination of suitable linkers between the individual DNA-bindingdomains of the protein. See, also, U.S. Publication No. 20110301073.

Donors

As noted above, insertion of an exogenous sequence (also called a “donorsequence” or “donor” or “transgene”), for example, for expression of apolypeptide, correction of a mutant gene or for increased expression ofa wild-type gene. It will be readily apparent that the donor sequence istypically not identical to the genomic sequence where it is placed. Adonor sequence can contain a non-homologous sequence flanked by tworegions of homology to allow for efficient HDR at the location ofinterest. Additionally, donor sequences can comprise a vector moleculecontaining sequences that are not homologous to the region of interestin cellular chromatin. A donor molecule can contain several,discontinuous regions of homology to cellular chromatin. For example,for targeted insertion of sequences not normally present in a region ofinterest, said sequences can be present in a donor nucleic acid moleculeand flanked by regions of homology to sequence in the region ofinterest.

The donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. See, e.g., U.S. Patent Publication Nos. 20100047805; 20110281361;20110207221 and 20130326645. The donor sequence(s) can be containedwithin a DNA MC, which may be introduced into the cell in circular orlinear form. If introduced in linear form, the ends of the donorsequence can be protected (e.g., from exonucleolytic degradation) bymethods known to those of skill in the art. For example, one or moredideoxynucleotide residues are added to the 3′ terminus of a linearmolecule and/or self-complementary oligonucleotides are ligated to oneor both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad.Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.Additional methods for protecting exogenous polynucleotides fromdegradation include, but are not limited to, addition of terminal aminogroup(s) and the use of modified internucleotide linkages such as, forexample, phosphorothioates, phosphoramidates, and O-methyl ribose ordeoxyribose residues.

A polynucleotide can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover,donor polynucleotides can be introduced as naked nucleic acid, asnucleic acid complexed with an agent such as a liposome or poloxamer, orcan be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

The donor is generally inserted so that its expression is driven by theendogenous promoter at the integration site, namely the promoter thatdrives expression of the endogenous gene into which the donor isinserted (e.g., AAVS1, CCR5, HPRT etc. (see co-owned US patent U.S. Pat.Nos. 8,110,379 and 7,951,925, and U.S. Publication Nos. 20130137104 and20130122591). However, it will be apparent that the donor may comprise apromoter and/or enhancer, for example a constitutive promoter or aninducible or tissue specific promoter.

Targeted insertion of non-coding nucleic acid sequence may also beachieved. Sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs(miRNAs) may also be used for targeted insertions.

The donor molecule may be inserted into an endogenous gene such thatall, some or none of the endogenous gene is expressed. For example, atransgene as described herein may be inserted into an endogenous locussuch that some (N-terminal and/or C-terminal to the transgene) or noneof the endogenous sequences are expressed, for example as a fusion withthe transgene. In other embodiments, the transgene (e.g., with orwithout additional coding sequences such as for the endogenous gene) isintegrated into any endogenous locus, for example a safe-harbor locus.See, e.g., U.S. patent publications 20080299580; 20080159996 and201000218264.

When endogenous sequences (endogenous or part of the transgene) areexpressed with the transgene, the endogenous sequences may befull-length sequences (wild-type or mutant) or partial sequences.Preferably the endogenous sequences are functional. Non-limitingexamples of the function of these full length or partial sequencesinclude increasing the serum half-life of the polypeptide expressed bythe transgene (e.g., therapeutic gene) and/or acting as a carrier.

Furthermore, although not required for expression, exogenous sequencesmay also include transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

In certain embodiments, the exogenous sequence (donor) comprises afusion of a protein of interest and, as its fusion partner, anextracellular domain of a membrane protein, causing the fusion proteinto be located on the surface of the cell. In some instances, the donorencodes a CAR wherein the CAR encoding sequences are inserted into asafe harbor such that the CAR is expressed. In some instances, the CARencoding sequences are inserted into a PD1 and/or a CTLA-4 locus. Inother cases, the CAR is delivered to the cell in a lentivirus for randominsertion while the PD1- or CTLA-4 specific nucleases are supplies asmRNAs. In some instances, the CAR is delivered via a viral vector systemsuch as AAV or adenovirus along with mRNA encoding nucleases specificfor a safe harbor (e.g. AAVS1, CCR5, albumin or HPRT). See, U.S. PatentPublication Nos. 20080299580; 20080159996; 201000218264; 20110301073;20130177983 and 20130177960 and 20150056705. The cells can also betreated with mRNAs encoding PD1 and/or CTLA-4 specific nucleases. Incertain embodiments, the polynucleotide encoding the CAR is supplied viaa viral delivery system together with mRNA encoding HPRT specificnucleases and PD1- or CTLA-4 specific nucleases. Cells comprising anintegrated CAR-encoding nucleotide at the HPRT locus can be selected forusing 6-thioguanine, a guanine analog that can result in cell arrestand/or initiate apoptosis in cells with an intact HPRT gene. CARs thatcan be used with the methods and compositions of the invention includeall types of these chimeric proteins, including first, second and thirdgeneration designs. CARS comprising specificity domains derived fromantibodies are particularly useful, although specificity domains derivedfrom receptors, ligands and engineered polypeptides are also envisionedby the invention. The intercellular signaling domains can be derivedfrom TCR chains such as zeta and other members of the CD3 complex suchas the γ and ε chains. In some cases, the CARs may comprise additionalco-stimulatory domains such as the intercellular domains from CD28,CD137 (also known as 4-1BB) or CD134. In still further cases, two typesof co-stimulator domains may be used simultaneously (i.e. CD3 zeta usedwith CD28+CD137).

Fusion Proteins

Fusion proteins comprising DNA-binding proteins (e.g., ZFPs or TALEs) asdescribed herein and a heterologous regulatory (functional) domain (orfunctional fragment thereof) are also provided. Common domains include,e.g., transcription factor domains (activators, repressors,co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun,fos, myb, max, mad, rel, ets, bel, myb, mos family members etc.); DNArepair enzymes and their associated factors and modifiers; DNArearrangement enzymes and their associated factors and modifiers;chromatin associated proteins and their modifiers (e.g. kinases,acetylases and deacetylases); and DNA modifying enzymes (e.g.,methyltransferases, topoisomerases, helicases, ligases, kinases,phosphatases, polymerases, endonucleases) and their associated factorsand modifiers. U.S. Patent Publication Nos. 20050064474; 20060188987 and2007/0218528 for details regarding fusions of DNA-binding domains andnuclease cleavage domains, incorporated by reference in their entiretiesherein.

Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factorkappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Beerliet al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron(Molinari et al., (1999) EMBO J. 18, 6439-6447). Additional exemplaryactivation domains include, Oct 1, Oct-2A, Sp 1, AP-2, and CTF1 (Seipelet al., EMBO J. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol.Endocrinol. 14:329-347; Collingwood et al. (1999) J Mol. Endocrinol.23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska(1999) Acta Biochim. Pol. 46:77-89; McKenna et al. (1999) J. SteroidBiochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci.25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504.Additional exemplary activation domains include, but are not limited to,OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP/GP,and TRAB1. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanamiet al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev.5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al.(1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al.(2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44;and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

It will be clear to those of skill in the art that, in the formation ofa fusion protein (or a nucleic acid encoding same) between a DNA-bindingdomain and a functional domain, either an activation domain or amolecule that interacts with an activation domain is suitable as afunctional domain. Essentially any molecule capable of recruiting anactivating complex and/or activating activity (such as, for example,histone acetylation) to the target gene is useful as an activatingdomain of a fusion protein. Insulator domains, localization domains, andchromatin remodeling proteins such as ISWI-containing domains and/ormethyl binding domain proteins suitable for use as functional domains infusion molecules are described, for example, in co-owned U.S. PatentPublications 2002/0115215 and 2003/0082552 and in co-owned WO 02/44376.

Exemplary repression domains include, but are not limited to, KRAB A/B,KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3,members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2.See, for example, Bird et al. (1999) Cell 99:451-454; Tyler et al.(1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; andRobertson et al. (2000) Nature Genet. 25:338-342. Additional exemplaryrepression domains include, but are not limited to, ROM2 and AtHD2A.See, for example, Chem et al. (1996) Plant Cell 8:305-321; and Wu et al.(2000) Plant J. 22:19-27.

The functional component/domain of a fusion molecule can be selectedfrom any of a variety of different components capable of influencingtranscription of a gene once the fusion molecule binds to a targetsequence via its DNA binding domain. Hence, the functional component caninclude, but is not limited to, various transcription factor domains,such as activators, repressors, co-activators, co-repressors, andsilencers.

Additional exemplary functional domains are disclosed, for example, inco-owned U.S. Pat. No. 6,534,261 and US Patent Publication No.2002/0160940.

Functional domains that are regulated by exogenous small molecules orligands may also be selected. For example, RheoSwitch® technology may beemployed wherein a functional domain only assumes its activeconformation in the presence of the external RheoChem™ ligand (see forexample US 20090136465). Thus, the ZFP, TALE or Cas may be operablylinked to the regulatable functional domain wherein the resultantactivity of the ZFP-TF, TALE-TF or CRISPR/Cas TF is controlled by theexternal ligand.

Nucleases

In certain embodiments, the fusion protein comprises a DNA-bindingbinding domain and cleavage (nuclease) domain. As such, genemodification can be achieved using a nuclease, for example an engineerednuclease. Engineered nuclease technology is based on the engineering ofnaturally occurring DNA-binding proteins. The methods and compositionsdescribed herein are broadly applicable and may involve any nuclease ofinterest. Non-limiting examples of nucleases include meganucleases,TALENs, zinc finger nucleases, and CRISPR/Cas nuclease systems. Thenuclease may comprise heterologous DNA-binding and cleavage domains(e.g., zinc finger nucleases; TALENs, meganuclease DNA-binding domainswith heterologous cleavage domains) or, alternatively, the DNA-bindingdomain of a naturally-occurring nuclease may be altered to bind to aselected target site (e.g., a meganuclease that has been engineered tobind to site different than the cognate binding site). For example,engineering of homing endonucleases with tailored DNA-bindingspecificities has been described, see, Chames et al. (2005) NucleicAcids Res 33(20):e178; Arnould et al. (2006) J. Mol. Biol. 355:443-458and Grizot et al (2009) Nucleic Acids Res July 7 e publication. Inaddition, engineering of ZFPs has also been described. See, e.g., U.S.Pat. Nos. 6,534,261; 6,607,882; 6,824,978; 6,979,539; 6,933,113;7,163,824; and 7,013,219. The nuclease may comprise combinations ofnucleic acid and protein (e.g., CRISPR/Cas).

In certain embodiment, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG (SEQ ID NO: 121) family, the GIY-YIG family, the His-Cystbox family and the HNH family. Exemplary homing endonucleases includeI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Theirrecognition sequences are known. See also U.S. Pat. No. 5,420,032; U.S.Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res.25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994)Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228;Gimble et al. (1996)J Mol. Biol. 263:163-180; Argast et al. (1998) J.Mol. Biol. 280:345-353 and the New England Biolabs catalogue.

DNA-binding domains from naturally-occurring meganucleases, primarilyfrom the LAGLIDADG (SEQ ID NO: 121) family, have been used to promotesite-specific genome modification in plants, yeast, Drosophila,mammalian cells and mice, but this approach has been limited to themodification of either homologous genes that conserve the meganucleaserecognition sequence (Monet et al. (1999), Biochem. Biophysics. Res.Common. 255: 88-93) or to pre-engineered genomes into which arecognition sequence has been introduced (Route et al. (1994), Mol.Cell. Biol. 14: 8096-106; Chilton et al. (2003), Plant Physiology. 133:956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93: 5055-60;Rong et al. (2002), Genes Dev. 16: 1568-81; Gouble et al. (2006), J.Gene Med. 8(5):616-622). Accordingly, attempts have been made toengineer meganucleases to exhibit novel binding specificity at medicallyor biotechnologically relevant sites (Porteus et al. (2005), Nat.Biotechnol. 23: 967-73; Sussman et al. (2004), J Mol. Biol. 342: 31-41;Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62; Chevalier et al.(2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic AcidsRes.31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques etal. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication Nos.20070117128; 20060206949; 20060153826; 20060078552; and 20040002092). Inaddition, naturally-occurring or engineered DNA-binding domains frommeganucleases have also been operably linked with a cleavage domain froma heterologous nuclease (e.g., FokI).

In other embodiments, the nuclease is a zinc finger nuclease (ZFN). ZFNscomprise a zinc finger protein that has been engineered to bind to atarget site in a gene of choice and cleavage domain or a cleavagehalf-domain.

As noted above, zinc finger binding domains can be engineered to bind toa sequence of choice. See, for example, Beerli et al. (2002) NatureBiotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol. 19: 656-660; Segalet al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000)Curr. Opin. Struct. Biol. 10:411-416. An engineered zinc finger bindingdomain can have a novel binding specificity, compared to anaturally-occurring zinc finger protein. Engineering methods include,but are not limited to, rational design and various types of selection.Rational design includes, for example, using databases comprisingtriplet (or quadruplet) nucleotide sequences and individual zinc fingeramino acid sequences, in which each triplet or quadruplet nucleotidesequence is associated with one or more amino acid sequences of zincfingers which bind the particular triplet or quadruplet sequence. See,for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261,incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; ZFNs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. PatentPublication Nos. 20050064474 and 20060188987, incorporated by referencein their entireties herein.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, e.g., U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

In some embodiments, the nuclease is an engineered TALEN. Methods andcompositions for engineering these proteins for robust, site specificinteraction with the target sequence of the user's choosing have beenpublished (see co-owned US patent Publication No. 20110301073).

In other embodiments, the nuclease is a CRISPR/Cas nuclease system asdescribed herein.

Nucleases such as ZFNs, TALENs, CRISPR/Cas and/or meganucleases alsocomprise a nuclease (cleavage domain, cleavage half-domain). As notedabove, the cleavage domain may be homologous or heterologous to theDNA-binding domain. For example, cleavage domains can include Casnucleases (in a CRISPR/Cas system) or meganuclease cleavage domains witha meganuclease DNA-binding domain. Alternatively, heterologous cleavagedomains include fusions proteins comprising zinc finger or TALEDNA-binding domain and a cleavage domain from a nuclease or ameganuclease DNA-binding domain and cleavage domain from a differentnuclease. Heterologous cleavage domains can be obtained from anyendonuclease or exonuclease. Exemplary endonucleases from which acleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31, 978-982. Thus, in one embodiment, fusionproteins comprise the cleavage domain (or cleavage half-domain) from atleast one Type IIS restriction enzyme and one or more zinc fingerbinding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95:10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I or TALE-FokI fusions, two fusionproteins, each comprising a FokI cleavage half-domain, can be used toreconstitute a catalytically active cleavage domain. Alternatively, asingle polypeptide molecule containing a zinc finger or TALE DNA bindingdomain and two Fok I cleavage half-domains can also be used. Parametersfor targeted cleavage and targeted sequence alteration using zincfinger- or TALE-Fok I fusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987 and20080131962, the disclosures of all of which are incorporated byreference in their entireties herein. Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of FokI are all targets for influencing dimerizationof the FokI cleavage half-domains.

Exemplary engineered cleavage half-domains of FokI that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFokI and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., Example1 of co-owned U.S. Patent publication No. 20080131962, and issued U.S.Pat. No. 7,914,796, the disclosures of which are incorporated byreference in their entirety for all purposes.

In certain embodiments, the engineered cleavage half-domain comprisesmutations at positions 486, 499 and 496 (numbered relative to wild-typeFokI), for instance mutations that replace the wild type Gln (Q) residueat position 486 with a Glu (E) residue, the wild-type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See, U.S. Patent Publication No. 20110201055).Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474; 20080131962; and 20110201055.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) in U.S. Patent Publication Nos.20050064474; 2009/0305346; 2008/0131962; and 20110201055.

Nuclease expression constructs can be readily designed using methodsknown in the art. See, e.g., United States Patent Publications20030232410; 20050208489; 20050026157; 20050064474; 20060188987;20060063231; and International Publication WO 07/014275. In certainembodiments, expression of the nuclease is under the control of aninducible promoter, for example the galactokinase promoter which isactivated (de-repressed) in the presence of raffinose and/or galactoseand repressed in presence of glucose. In particular, the galactokinasepromoter is induced and the nuclease(s) expressed upon successivechanges in the carbon source (e.g., from glucose to raffinose togalactose). Other non-limiting examples of inducible promoters includeCUP1, MET15, PHO5, and tet-responsive promoters.

Nucleases that generate single-stranded breaks can also be used. Incertain embodiments, a catalytically inactive nuclease is used incombination with a catalytically active nuclease to generate asingle-stranded break (also referred to as “nickases”). Such nickasesare described, for example, in U.S. Patent Publication No. 20100047805;Jinek et al, ibid; Cong et al., ibid. Nickases can be generated byspecific mutation of amino acids in the catalytic domain of the enzyme,or by truncation of part or all of the domain such that it is no longerfunctional. Thus, in nucleases comprising two nuclease (cleavage)domains (e.g., ZFNs, TALENs, and CRISPR/Cas nuclease systems), thisapproach may be taken on either domain. Furthermore, a double strandbreak can be achieved in the target DNA by the use of two suchsingle-stranded nickases. Each nickase cleaves one strand of the DNA andthe use of two or more nickases can create a double strand break (e.g.,a staggered double-stranded break) in a target double-stranded sequence.

Delivery

The nucleases and transcription factors, polynucleotides encoding same,and/or any donor polynucleotides and compositions comprising theproteins and/or polynucleotides described herein may be delivered to atarget cell by any suitable means.

Suitable cells include but not limited to eukaryotic and prokaryoticcells and/or cell lines. Non-limiting examples of such cells or celllines generated from such cells include COS, CHO (e.g., CHO-S, CHO-K1,CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79,B14AF28-G3, BHK, HaK, NSO, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F,HEK293-H, HEK293-T), and perC6 cells as well as insect cells such asSpodoptera frugiperda (Sf), or fungal cells such as Saccharomyces,Pichia and Schizosaccharomyces. In certain embodiments, the cell line isa CHO-K1, MDCK or HEK293 cell line. Suitable primary cells includeperipheral blood mononuclear cells (PBMC), and other blood cell subsetssuch as, but not limited to, any T-cell, such as CD4+ T-cells, CD8+T-cells, tumor infiltrating cells (TILs) or any other type of T-cell.Suitable cells also include stem cells such as, by way of example,embryonic stem cells, induced pluripotent stem cells, hematopoietic stemcells, neuronal stem cells and mesenchymal stem cells.

Methods of delivering transcription factors and nucleases as describedherein are described, for example, in U.S. Pat. Nos. 6,453,242;6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978;6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of allof which are incorporated by reference herein in their entireties.

The transcription factors and nucleases as described herein may also bedelivered using vectors, for example containing sequences encoding oneor more of the proteins. Donor encoding polynucleotides may be similarlydelivered. Any vector systems may be used including, but not limited to,plasmid vectors, retroviral vectors, lentiviral vectors, adenovirusvectors, poxvirus vectors; herpesvirus vectors and adeno-associatedvirus vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporatedby reference herein in their entireties. Furthermore, it will beapparent that any of these vectors may comprise one or moretranscription factor and/or nuclease. Thus, when one or more ZFPs,TALEs, CRISPR/Cas molecules and/or donors are introduced into the cell,the ZFPs, TALEs, CRISPR/Cas molecules and/or donors may be carried onthe same vector or on different vectors. When multiple vectors are used,each vector may comprise a sequence encoding one or multiple ZFPs,TALEs, CRISPR/Cas molecules and/or donors.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered ZFPs, CRISPR/Casmolecules, TALEs and/or donors in cells (e.g., mammalian cells) andtarget tissues. Such methods can also be used to administer nucleicacids encoding ZFPs, TALES, CRISPR/Cas molecules, and/or donors to cellsin vitro. In certain embodiments, nucleic acids encoding ZFPs, TALEs,CRISPR/Cas molecules, and/or donors are administered for in vivo or exvivo gene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome or poloxamer. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel&Felgner,TIBTECH 11:211-217 (1993); Mitani&Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology, Doerfler and Bohm (eds.) (1995); and Yuet al., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,mRNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa® Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No.4,946,787; and U.S. Pat. No. 4,897,355) and lipofection reagents aresold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al (2009) Nature Biotechnology 27(7) p. 643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs, TALEs, CRISPR/Cas molecules, and/ordonors take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery include, but are not limited to, retroviral,lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplexvirus vectors for gene transfer. Integration in the host genome ispossible with the retrovirus, lentivirus, and adeno-associated virusgene transfer methods, often resulting in long term expression of theinserted transgene. Additionally, high transduction efficiencies havebeen observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors is described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type virus. The vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,AAV6 and AAV8, AAV 8.2, AAV9, and AAV rh10 and pseudotyped AAV such asAAV2/8, AAV2/5 and AAV2/6 can also be used in accordance with thepresent invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, AAV, and ψ2 cells or PA317 cells, which package retrovirus.Viral vectors used in gene therapy are usually generated by a producercell line that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionally,AAV can be produced at clinical scale using baculovirus systems (seeU.S. Pat. No. 7,479,554.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFPnucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

Suitable cells include but not limited to eukaryotic and prokaryoticcells and/or cell lines. Non-limiting examples of such cells or celllines generated from such cells include COS, CHO (e.g., CHO-S, CHO-K1,CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79,B14AF28-G3, BHK, HaK, NSO, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F,HEK293-H, HEK293-T), and perC6 cells as well as insect cells such asSpodoptera frugiperda (Sf), or fungal cells such as Saccharomyces,Pichia and Schizosaccharomyces. In certain embodiments, the cell line isa CHO-K1, MDCK or HEK293 cell line. Additionally, primary cells may beisolated and used ex vivo for reintroduction into the subject to betreated following treatment with the transcription factors and/ornucleases described herein. Suitable primary cells include peripheralblood mononuclear cells (PBMC), and other blood cell subsets such as,but not limited to, T-cells such as tumor infiltrating cells (TILs),CD4+ T-cells or CD8+ T-cells. Suitable cells also include stem cellssuch as, by way of example, embryonic stem cells, induced pluripotentstem cells, hematopoietic stem cells, neuronal stem cells andmesenchymal stem cells.

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see, Inaba et al., J. Exp.Med. 176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T-cells), CD45+ (panBcells), GR-1 (granulocytes),and Tad (differentiated antigen presenting cells) (see, Inaba et al., J.Exp. Med. 176:1693-1702 (1992)).

Stem cells that have been modified may also be used in some embodiments.For example, stem cells that have been made resistant to apoptosis maybe used as therapeutic compositions where the stem cells also containthe ZFPs, TALEs, CRISPR/Cas molecules and/or donors of the invention.Resistance to apoptosis may come about, for example, by knocking out BAXand/or BAK using BAX- or BAK-specific ZFNs (see, U.S. Pat. No.8,597,912) in the stem cells, or those that are disrupted in a caspase,again using caspase-6 specific ZFNs for example.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP, TALE, CRISPR/Cas molecules and/or donor nucleic acidscan also be administered directly to an organism for transduction ofcells in vivo. Alternatively, naked DNA or mRNA can be administered.Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells including, butnot limited to, injection, infusion, topical application andelectroporation. Suitable methods of administering such nucleic acidsare available and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

Applications

The disclosed compositions and methods can be used for any applicationin which it is desired to modulate the expression of PD-land/or CTLA-4.In particular, these methods and compositions can be used wheremodulation of PD-1 or CTLA-4 is desired, including but not limited to,therapeutic and research applications. The invention also contemplatesinsertion of DNA sequences encoding a CAR and/or an engineered TCR intothe genome of the PD-1 and/or CTLA-4 modulated cells (e.g., cells inwhich PD1 and/or CTLA-4 expression is modified via an engineeredtranscription factor or is knocked out using engineered nucleases). Insome instances, the cells are TILs or cells expanded from TILs. Themethods and compositions may be used to treat various disease anddisorders including chronic infectious diseases such as HIV/AIDS and HCVand/or cancers (e.g., melanoma, ovarian cancer, colorectal/colon cancer,renal cell carcinoma, plasmacytoma/myeloma, breast cancer and lungcancer).

These and other diseases may also be treated with PD1- or CTLA-4targeting nucleases or transcription factors in combination with CARswherein the CARs are introduced into the cell via a viral deliverysystem. In some cases, an engineered TCR is also introduced into thecell, or may be introduced into the cell instead of a CAR. To facilitateoperation of the engineered TCR, the endogenous TCR may also bedisrupted.

Methods and compositions comprising PD1- or CTLA-4 specific nucleases ortranscription factors may also be used in conjunction with othertherapeutics designed to treat a chronic infectious disease or cancer.The nucleases as described herein (e.g., ZFNs, TALENs, CRISPR/Cassystems or polynucleotides encoding these molecules) or transcriptionfactors (or polynucleotides encoding them) may be administeredconcurrently (e.g., in the same pharmaceutical compositions) or may beadministered sequentially in any order. Any type of cancer can betreated, including, but not limited to lung carcinomas, pancreaticcancers, liver cancers, bone cancers, breast cancers, colorectalcancers, ovarian cancers, leukemias, melanomas, lymphomas, brain cancersand the like.

The PD1 and/or CTLA-4 specific nucleases or transcription factors may beused in conjunction with a CAR T-cell targeting system. The CARs mayhave specificity for a tumor antigen where the CAR specificity domain isa ScFv. Alternatively, CARs may be specific for a tumor antigen wherethe CAR specificity domain comprises a ligand or polypeptide.Non-limiting exemplary CARs include those targeted to CD33 (see Dutouret al, (2012) Adv Hematol 2012; 2012:683065), GD2 (Louis et al (2011)Blood 118(23):650-6), CD19 (Savoldo et al, (2011) J Clin Invest 121(5):1822 and Torikai et al (2012) Blood 119(24): 5697), IL-11Rα (Huang etal, (2012) Cancer Res 72(1):271-81), CD20 (Till et al (2012) Blood119(17):3940-50), NY-ESO-1 (Schuberth et al, (2012) Gene Therdoi:10.1038/gt2012.48), ErbB2 (Zhao et al, (2009) J. Immunol 183(9):5563-74), CD70 (Shaffer et al (2011) Blood 116(16):4304-4314), CD38(Bhattacharayya et al (2012) Blood Canc J 2(6) p. e75), CD22 (Haso etal. (2012) Canc Res 72(8) S1, doi: 1158/1158-7445 AM2012-3504), CD74(Stein et al (2004) Blood 104:3705-3711), CAIX (Lamers et al, (2011)Blood 117(1): 72-82) STEAP1 (see Kiessling et al. (2012) Cancers4:193-217 for review of target) VEGF-R2 (U.S. Patent Publication No.US20120213783A1), the folate receptor (PCT patent publicationWO2012099973) and IL-13 Rα (U.S. Pat. No. 7,514,537). In some cases, theCAR may be bi-specific (see US Patent publication No. US2001012967). Insome cases, the T-cells are TILs. Additionally, the PD1 and/or CTLA-4specific nucleases or transcription factors may be used in conjunctionwith a T cell or TIL comprising an engineered TCR.

The methods and compositions of the invention are also useful for thedesign and implementation of in vitro and in vivo models, for example,animal models of chronic infection, cancer or autoimmunity, which allowsfor the study of these disorders and furthers discovery of usefultherapeutics. In some cases, the methods of the invention are useful forproducing engineered T-cells that may be used in patients in needthereof. For some treatments, the patients are pretreated with agentsfor partial or full myoablation prior to infusion of the T-cells.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a ZFN or TALEN. It will beappreciated that this is for purposes of exemplification only and thatother nucleases can be used, for instance CRISPR/Cas nuclease systems,homing endonucleases (meganucleases) with engineered DNA-binding domainsand/or fusions of naturally occurring of engineered homing endonucleases(meganucleases) DNA-binding domains and heterologous cleavage domains.

EXAMPLES Example 1: Identification of Persistently Biologically ActivePD1- or CTLA-4 Specific ZFNs

ZFNs were assembled against the human PD1 or CTLA-4 genes and weretested by ELISA and CEL1 assays as described in Miller et al. (2007)Nat. Biotechnol. 25:778-785 and U.S. Patent Publication No. 20050064474and International Patent Publication WO2005/014791.

Specific examples of PD1-targeted ZFPs are disclosed in U.S. PatentPublication No. 20110136895 and shown in Table 2a and 2b andCTLA-4-targeted ZFP designs are shown in Table 2c. The first column inthis table is an internal reference name (number) for a ZFP. “F” refersto the finger and the number following “F” refers to which zinc finger(e.g., “F1” refers to finger 1). The target sites for these CTLA-4specific ZFNs are shown in Table 3.

TABLE 2a Human PD1-targeted zinc finger proteins Design SBS # F1 F2 F3F4 F5 F6 12942 QSGHLSR RSDSLSV HNDSRKN RSDDLTR RSDHLTQ N/A (SEQ ID (SEQID (SEQ ID (SEQ ID (SEQ ID NO: 34) NO: 35) NO: 36) NO: 37) NO: 38) 12946RSAALSR RSDDLTR RSDHLTT DRSALSR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQID (SEQ ID NO: 39) NO: 37) NO: 40) NO: 6) NO: 41) 12947 RSAALAR RSDDLSKRNDHRKN DRSALSR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:42) NO: 3) NO: 43) NO: 6) NO: 41) 12934 RSDHLSE TSSDRTK RSDHLSE QSASRKNN/A N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 44) NO: 45) NO: 44) NO: 46)12971 RSDVLSE RSANLTR RSDHLSQ TSSNRKT DRSNLSR RSDALAR (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 47) NO: 48) NO: 49) NO: 50) NO: 9)NO: 7) 12972 DDWNLSQ RSANLTR RSDHLSQ TSSNRKT DRSNLSR RSDALAR (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 51) NO: 48) NO: 49) NO: 50)NO: 9) NO: 7) 18759 RSSALSR RPLALKH RNDHRKN TRPVLKR DRSALAR N/A (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 52) NO: 53) NO: 43) NO: 54) NO: 41)22237 QSGHLSR RSDSLSV HNDSRKN RANSLLR RSDHLTQ N/A (SEQ ID (SEQ ID (SEQID (SEQ ID (SEQ ID NO: 34) NO: 35) NO: 36) NO: 55) NO: 38) 25005 RPSTLHRRSDELTR RNNNLRT TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQID NO: 56) NO: 57) NO: 58) NO: 54) NO: 41) 25006 RPSTLHR RSDELTR TNWHLRTTRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 56) NO:57) NO: 59) NO: 54) NO: 41) 25010 RPSTLHR RSDELTR RTPHLTL TRPVLKRDRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 56) NO: 57) NO:60) NO: 54) NO: 41) 25011 RPSTLHR RSDELTR RSAQLAT TRPVLKR DRSALAR N/A(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 56) NO: 57) NO: 61) NO: 54)NO: 41) 25012 RPSTLHR RSDELTR RCTHLYL TRPVLKR DRSALAR N/A (SEQ ID (SEQID (SEQ ID (SEQ ID (SEQ ID NO: 56) NO: 57) NO: 62) NO: 54) NO: 41) 25013RPSTLHR RSDELTR RPTQRYS TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQID (SEQ ID NO: 56) NO: 57) NO: 63) NO: 54) NO: 41) 25014 RPSTLHR RSDELTRRANHREC TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:56) NO: 57) NO: 64) NO: 54) NO: 41) 25015 RPSTLHR RSDELTR RANHRECTRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 56) NO:57) NO: 64) NO: 54) NO: 41) 25016 RKFARPS RNFSRSD HPHHRMC TRPVLKRDRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 65) NO: 66) NO:67) NO: 54) NO: 41) 25017 RPSTLHR RSDELTR RMGRLST TRPVLKR DRSALAR N/A(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 56) NO: 57) NO: 68) NO: 54)NO: 41) 25022 RPSTLHR RSDELTR RHSRLTT TRPVLMR DRSALAR N/A (SEQ ID (SEQID (SEQ ID (SEQ ID (SEQ ID NO: 56) NO: 57) NO: 69) NO: 70) NO: 41) 25023RPSTLHR RSDELTR RANHRVC TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQID (SEQ ID NO: 56) NO: 57) NO: 71) NO: 54) NO: 41) 25025 RPSTLHR RSDELTRRSTHLLG TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:56) NO: 57) NO: 72) NO: 54) NO: 41) 25027 RNAALTR RSDELTR RSCGLWSTRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 73) NO:57) NO: 74) NO: 54) NO: 41) 25028 CNAALTR RSDELTR REEHRAT TRPVLKRDRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 75) NO: 57) NO:76) NO: 54) NO: 41) 25029 RNAALTR RSDELTR RHHHLAA TRPVLKR DRSALAR N/A(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 73) NO: 57) NO: 77) NO: 54)NO: 41) 25030 RNAALTR RSDELTR RPMHLTN TRPVLKR DRSALAR N/A (SEQ ID (SEQID (SEQ ID (SEQ ID (SEQ ID NO: 73) NO: 57) NO: 78) NO: 54) NO: 41) 25031RNAALTR RSDELTR RSPHLYH TRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQID (SEQ ID NO: 73) NO: 57) NO: 79) NO: 54) NO: 41) 25032 RNAALTR RSDELTRRCEALFIH TRPVLKR DRSAQAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:73) NO: 57) NO: 80) NO: 54) NO: 81) 25034 RNAALTR RSDELTR RCEALFIHTRPVLKR DRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 73) NO:57) NO: 80) NO: 54) NO: 41) 25036 RNAALTR RSDELTR RSPHLYH TRPVLKRDRSALAR N/A (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 73) NO: 57) NO:79) NO: 54) NO: 41) 25040 RNAALTR RSDELTR RLPALLS TRPVLKR DRSALAR N/A(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 73) NO: 57) NO: 82) NO: 54)NO: 41) 25041 HNAALTR RSDELTR RTYNRTQ TRPVLKR DRSALAR N/A (SEQ ID (SEQID (SEQ ID (SEQ ID (SEQ ID NO: 83) NO: 57) NO: 84) NO: 54) NO: 41)

TABLE 2b ZFN Target sites in the human PD1 gene SBS# Target site 12942ccAGGGCGCCTGTGGGAtctgcatgcct (SEQ ID NO: 85) 12946caGTCGTCTGGGCGGTGctacaactggg (SEQ ID NO: 86) 12947caGTCGTCTGGGCGGTGctacaactggg (SEQ ID NO: 86) 12934gaACACAGGCACGGctgaggggtcctcc (SEQ ID NO: 87) 12971ctGTGGACTATGGGGAGCTGgatttcca (SEQ ID NO: 88) 12972ctGTGGACTATGGGGAGCTGgatttcca (SEQ ID NO: 88) 18759 caGTCGTCTGGGCGGTGct(SEQ ID NO: 89) 22237 ccAGGGCGCCTGTGGGAtctgcatgcct (SEQ ID NO: 85) 25005caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25006 caGTCGTCTGGGCGGTGct (SEQ IDNO: 89) 25010 caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25011caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25012 caGTCGTCTGGGCGGTGct (SEQ IDNO: 89) 25013 caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25014caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25015 caGTCGTCTGGGCGGTGct (SEQ IDNO: 89) 25016 caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25017caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25022 caGTCGTCTGGGCGGTGct (SEQ IDNO: 89) 25023 caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25025caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25027 caGTCGTCTGGGCGGTGct (SEQ IDNO: 89) 25028 caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25029caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25030 caGTCGTCTGGGCGGTGct (SEQ IDNO: 89) 25031 caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25032caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25034 caGTCGTCTGGGCGGTGct (SEQ IDNO: 89) 25036 caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25040caGTCGTCTGGGCGGTGct (SEQ ID NO: 89) 25041 caGTCGTCTGGGCGGTGct (SEQ IDNO: 89)

TABLE 2c Human CTLA-4-targeted zinc finger proteins Design SBS # F1 F2F3 F4 F5 20186 QSSDLSR RSDNLRE RSDDLSK QSSDLRR LKQHLNE (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 1) NO: 2) NO: 3) NO: 4) NO: 5) 20185 DRSALSRRSDALAR QSGDRNK DRSNLSR RSDDRKT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 6) NO: 7) NO: 8) NO: 9) NO: 10) 20190 QSGSLTR RSDNLTT QNATRIKRSDVLSA DRSNRIK (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 11) NO: 12)NO: 13) NO: 14) NO: 15) 20189 RSANLAR TNQNRIT TSGHLSR RSDSLLR RNDDRKK(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 16) NO: 17) NO: 18) NO: 19)NO: 20)

TABLE 3 ZFN Target sites in the human CTLA-4 genes SBS# Target site20186 acAGTGCTTCGgCAGGCTgacagccagg (SEQ ID NO: 21) 20185acCCGGACcTCAGTGGCTttgcctggag (SEQ ID NO: 22) 20190acTACCTGgGCATAGGCAacggaaccca (SEQ ID NO: 23) 20189tgGCGGTGGGTaCATGAGctccaccttg (SEQ ID NO: 24)

Initial in vitro activity assays were performed on nucleofected cellsamples as described above. Briefly, the plasmids encoding ZFP-FokIfusions were introduced into K562 cells by transfection using the Amaxa™Nucleofection kit as specified by the manufacturer. For transfection,two million K562 cells were mixed with varying amounts of eachzinc-finger nuclease expression plasmid and 1004, Amaxa™ Solution V.Cells were transfected in an Amaxa Nucleofector II™ using program T-16.Immediately following transfection, the cells were divided into twodifferent flasks and grown in RPMI medium (Invitrogen) supplemented with10% FBS in 5% CO₂ at either 30° C. or 37° C. for four days.

In addition, PMBCs were also activated with anti-CD28/CD38 beads (see,e.g., U.S. Publication No. 20080311095). Either 3 days (EPD3) or 5 days(EPD5) following activation, the cells were electroporated withPD1-specific ZFN mRNAs (PD1, particularly ZFNs 12942 and 25029) or CCR5(R5, see, U.S. Pat. No. 7,951,925) using two different MAXCYTE™conditions (C1 and C3). The cells were then analyzed for genemodification of the target locus using the CEL-1 assay described below.As shown in FIG. 2, high levels of gene modification was seen.

To determine the ZFN activity at the CTLA-4 locus, Cel-1 based SURVEYOR™Nuclease assays were performed essentially as per the manufacturer'sinstructions (Transgenomic SURVEYOR™) and as described for PD1 in U.S.Patent Publication No. 20110136895. Briefly, cells were harvested andchromosomal DNA prepared using a Quickextract™ Kit according tomanufacturer's directions (Epicentre®). The appropriate region of thePD1 locus was PCR amplified using Accuprime™ High-fidelity DNApolymerase (Invitrogen). PCR reactions were heated to 94° C., andgradually cooled to room temperature. Approximately 200 ng of theannealed DNA was mixed with 0.33 μL Cel-I enzyme and incubated for 20minutes at 42° C. Reaction products were analyzed by polyacrylamide gelelectrophoresis in 1× Tris-borate-EDTA buffer.

Cells were harvested 3 or 10 days after exposure to virus and genemodification efficiency was determined using a Cel-I based SURVEYOR′Nuclease assay, performed as described in International PatentPublication WO 07/014275. See, also, Oleykowski et al. (1998) NucleicAcids Res. 26:4597-4602; Qui et al. (2004) BioTechniques 36:702-707;Yeung et al. (2005) BioTechniques 38:749-758.

TABLE 4 Activity of CTLA-4 ZFNs ZFN pair % indels detected 20186/201853.3% 20190/20189 1.8%

Example 2: PD1 and CTLA-4 Specific TALENs

PD1 specific TALENs were developed and assembled as described previously(see US Patent Publication No. 20110301073). Base recognition wasachieved using the canonical RVD-base correspondences (the “TALE code”:NI for A, HD for C, NN for G (NK in half repeat), NG for T). The TALENswere constructed in the “+63” TALEN backbone as described in U.S.Publication No. 20110301073. The targets and numeric identifiers for theTALENs tested are shown below in Tables 5a and 5b.

TABLE 5a PD1 specific TALENs-Target site SBS # site # of RVDs SEQ ID NO:101621 gtAGCACCGCCCAGACGACtg 17 25 101618 gtGCTCCAGGCATGCAGATcc 17 26101620 atGCAGATCCCACAGGCgc 15 27 101622 gtTGTAGCACCGCCCAGACga 17 28101623 gtTGTAGCACCGCCCAGAcg 16 29 101624 atGCAGATCCCACAGGCgc 15 27101625 gtTGTAGCACCGCCCAGACga 17 28 101626 ctTCTCCCCAGCCCTGCTCgt 17 30101627 gtGAAGGTGGCGTTGTCCCct 17 31 101632 ctACCTCTGTGGGGCCATCtc 17 32101633 ctCTCTTTGATCTGCGCCTtg 17 33 101638 gtACCGCATGAGCCCCAGCaa 17 122101639 ctCGGGGAAGGCGGCCAGCtt 17 123 101640 ctACCTCTGTGGGGCCATCtc 17 32101641 ctCTCTTTGATCTGCGCCTtg 17 33

TABLE 5b PD1 specific TALENs-RVDs SEQ SBS # # of RVDs RVDs (N->C) ID NO:101621 17 NI-NN-HD-NI-HD-HD-NN-HD-HD- 90 HD-NI-NN-NI-HD-NN-NI-HD 10161817 NN-HD-NG-HD-HD-NI-NN-NN-HD- 91 NI-NG-NN-HD-NI-NN-NI-NG 101620 15NN-HD-NI-NN-NI-NG-HD-HD-HD- 92 NI-HD-NI-NN-NN-HD 101622 17NG-NN-NG-NI-NN-HD-NI-HD-HD- 93 NN-HD-HD-HD-NI-NN-NI-HD 101623 16NG-NN-NG-NI-NN-HD-NI-HD-HD- 94 NN-HD-HD-HD-NI-NN-NI 101624 15NN-HD-NI-NN-NI-NG-HD-HD-HD- 92 NI-HD-NI-NN-NN-HD- 101625 17NG-NN-NG-NI-NN-HD-NI-HD-HD- 93 NN-HD-HD-HD-NI-NN-NI-HD 101626 17NG-HD-NG-HD-HD-HD-HD-NI-NN- 95 HD-HD-HD-NG-NN-HD-NG-HD 101627 17NN-NI-NI-NN-NN-NG-NN-NN-HD- 96 NN-NG-NG-NN-NG-HD-HD-HD 101632 17NI-HD-HD-NG-HD-NG-NN-NG-NN- 97 NN-NN-NN-HD-HD-NI-NG-HD 101633 17HD-NG-HD-NG-NG-NG-NN-NI-NG- 98 HD-NG-NN-HD-NN-HD-HD-NG 101638 17NI-HD-HD-NN-HD-NI-NG-NN-NI- 99 NN-HD-HD-HD-HD-NI-NN-HD 101639 17HD-NN-NN-NN-NN-NI-NI-NN-NN- 100 HD-NN-NN-HD-HD-NI-NN-HD 101640 17NI-HD-HD-NG-HD-NG-NN-NG-NN- 97 NN-NN-NN-HD-HD-NI-NG-HD 101641 17HD-NG-HD-NG-NG-NG-NN-NI-NG- 98 HD-NG-NN-HD-NN-HD-ND-NG

The TALENs were then tested in pairs in K562 cells for the ability toinduce modifications at the endogenous PD1 chromosomal targets, and theresults showed that nearly all protein pairs were active. Side by sideactivity comparisons with the 12942/25029 ZFN pair (see U.S. PatentPublication No. 2011-136895) shown below in Table 6, showed that theTALENs and ZFNs have activities that are in the same approximate range.Note that the Lane numbers shown in Table 6 correspond to the lanesshown in FIG. 1.

TABLE 6 PD1 TALEN activity Lane TALEN pair % NHEJ 1 101621/101618 23.5 2101621/101619 18.1 3 101621/101620 0 4 101622/101618 14.7 5101622/101619 14.7 6 101622/101620 10.6 7 101623/101618 6.8 8101623/101619 18.2 9 101623/101620 11.6 10 101625/101624 10.1 1112942/25029 (ZFN) 14.7 G GFP 0 12 101627/101626 12.5 13 101633/10163214.7 14 101639/101638 0 15 101641/101640 23 G GFP 0

CTLA-4 specific TALENs are designed and assembled as described above.Testing for activity on the endogenous CTLA-4 chromosomal target revealsthat the TALENs are active.

Example 3: Generation of T-Cells Comprising a CAR that Also Lack PD1and/or CTLA-4

To generate a T-cell population that expresses a CAR and in which PD1and/or CTLA-4 are knocked out, CAR containing T-cells are generated.Cells (e.g., PBMCs, T-cells such as TILs, CD4+ or CD8+ cells) arepurified from natural sources, for example, a metastatic melanomapatient, and cultured and/or expanded according to standard procedures.Cells may be stimulated, for example, as described in U.S. PatentPublication No. 20080311095. Cells are transduced with CAR, for examplea CAR comprising either an ErbB2-specific scFv or a VEGFR2-specificscFv. The nucleic acids encoding the scFvs are first constructed via aPCR approach and are sequence verified. They are linked to CD28 and CD3zeta signaling moieties and introduced into the cells (e.g., viaretroviral or lentiviral or other targeting/delivery mechanisms).

The cells are then treated with mRNAs encoding PD1 and/orCTLA-4-specific nucleases and the population is analyzed by the Cel-Iassay to verify PD1 or CTLA-4 disruption and CAR insertion. Theengineered T-cells are then tested on tumor cell lines expressing eitherErbB2 or VEGF2R and shown to specifically lyse these target cell lines.

Example 4: PD1 Specific ZFP TFs

PD1 specific ZFP TFs were designed to repress expression of PD1expression. These proteins are shown below in Table 7a and 7b.

TABLE 7a Human PD1-targeted zinc finger proteins for ZFP-TFs Design SBS# F1 F2 F3 F4 F5 F6 22937 RSDTLSV DNSTRIK RSDHLSQ RSDVRKN DRSHLTRRSDNLTT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 101) NO:102) NO: 49) NO: 103) NO: 104) NO: 12) 22945 RSDDLTR RSDHLSR RSDNLARQSGNLAR RSDNLAR RSDALAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 37) NO: 105) NO: 106) NO: 107) NO: 106) NO: 7) 22954 QSGDLTR RSDDLTRRSDNLSV RSANLTR RSDVLSK QNATRIK (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 108) NO: 37) NO: 109) NO: 48) NO: 110) NO: 13) 22957 RSDVLSEARSTRTN DRSHLTR DRSHLAR QSGNLAR QSGHLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 47) NO: 111) NO: 104) NO: 112) NO: 107) NO: 34)22959 RSDNLSE DRSHLAR DRSHLTR QSSDLRR RSDHLST DRSNRKT (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 113) NO: 112) NO: 104) NO: 4) NO:114) NO: 115)

TABLE 7b Human PD1-targeted zinc finger proteins for ZFP-TFs, targetsites SBS# Target site 22937 ggTAGGGCGTGGGGGCCACGggcccacc_(SEQ ID NO:116) 22945 atGTGGAGGAAGAGGGGGCGggagcaag_(SEQ ID NO: 117) 22954gaGCAGTGGAGAAGGCGGCActctggtg_(SEQ ID NO: 118) 22957gtGGAGAAGGCGGCACTCTGgtggggct_(SEQ ID NO: 119) 22959acAACTGGGCTGGCGGCCAGgatggttc_(SEQ ID NO: 120)

The PD1-specific DNA binding domains depicted in Table 7a were thenfused to a KRAB repression domain from the human KOX1 gene. To test theactivity of the PD1 repressing ZFP TFs, the ZFP TFs were transfectedinto human cells and expression of PD1 was monitored using real-timeRT-PCR. Specifically, Jurkat cells were cultured in DMEM supplementedwith 10% FBS and 1e⁵ cells are transfected with 1 μg of plasmid DNAencoding indicated ZFP-KOX fusions by Amaxa Nucleofector® following themanufacturer's instructions.

Transfected cells were incubated for 2 days, and the levels ofendogenous human PD1 and normalization control 18S were analyzed byreal-time PCR (Applied Biosystems), according to standard protocols. PD1levels were expressed as PD1/18S ratios normalized to that of themock-transfected samples (set as 1).

PD1-Targeted ZFPs Repressed PD1 Expression.

Western blot analyses are done using standard protocols to confirm thereduction in PD1 protein level.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A genetically modified T-cell that expresses achimeric antigen receptor (CAR), wherein an exogenous sequence encodinga CAR is integrated into the genome of the T-cell, and further whereinexpression of at least one endogenous immunological checkpoint gene isrepressed by genetically modifying the immunological checkpoint gene inthe T-cell, wherein the immunological checkpoint gene is a programmeddeath receptor PDCD1 gene or a CTLA-4 gene.
 2. The T-cell of claim 1wherein the immunological checkpoint gene is a CTLA-4 gene.
 3. TheT-cell of claim 1, wherein the immunological checkpoint gene is PDCD1.4. The T-cell of claim 1, wherein the T-cell is selected from the groupconsisting of a CD4+ cell, a CD8+ cell and a tumor infiltrating cell(TIL).
 5. The T-cell of claim 1, wherein the exogenous sequence encodingthe CAR is integrated into the T-cell genome at a safe harbor locus. 6.The T-cell of claim 1, wherein the exogenous sequence encoding the CARis randomly integrated into the T-cell genome.
 7. The T-cell of claim 1,wherein the CAR comprises a signaling domain of a T-cell receptor (TCR).8. The T-cell of claim 7, wherein the CAR comprises a scFv specificitydomain.
 9. The T-cell of claim 1, further comprising at least oneadditional transgene.
 10. The T-cell of claim 9, wherein the at leastone additional transgene encodes a tumor-associated antigen(TAA)-specific T-cell receptor (TCR).
 11. The T-cell of claim 1, whereinthe T-cell is stimulated.
 12. The T-cell of claim 11, wherein the T-cellis stimulated with anti-CD28/CD3 beads.
 13. A method of making theT-cell of claim 5, the method comprising: cleaving the safe harbor geneand the at least one immunological checkpoint gene in the T-cell usingone or more nucleases such that the exogenous sequence encoding the CARis integrated into the safe harbor gene and the at least oneimmunological checkpoint gene is inactivated.
 14. The method of claim13, where the safe harbor gene is selected from the group consisting ofAAV51, CCR5, HPRT and Rosa.
 15. The method of claim 13, wherein theexogenous sequence encoding the CAR is carried by a plasmid vector or aviral vector.
 16. The method of claim 13, wherein the nucleases areintroduced into the cell as mRNA.
 17. The method of claim 13, whereinthe T-cell is selected from the group consisting of a CD4+ cell, a CD8+cell and a tumor infiltrating cell (TIL).
 18. The method of claim 13,wherein the CAR comprises a signaling domain of a T-cell receptor (TCR).19. The method of claim 13, wherein the CAR comprises a scFv specificitydomain.
 20. The method of claim 13, further comprising stimulating theT-cell.
 21. The method of claim 13, wherein the T-cell is stimulatedwith anti-CD28/CD3 beads.
 22. The method of claim 13, further comprisingintegrating at least additional transgene into the T-cell genome. 23.The method of claim 22, wherein the at least one additional transgeneencodes a TAA-specific T-cell receptor (TCR).
 24. A method of making theT-cell of claim 6, the method comprising: cleaving the at least oneimmunological checkpoint gene using one or more nucleases such that theat least one immunological checkpoint gene is inactivated; and randomlyintegrating the exogenous sequence encoding the CAR into the genome.